Composition and method for enhancing fibrinolysis

ABSTRACT

The present invention relates to a novel alpha-2-antiplasmin-binding molecules and treatment for pulmonary embolism, myocardial infarction, thrombosis or stroke in a patient which comprises administering an alpha-2-antiplasmin-binding molecule capable of preventing inhibition of plasmin by endogenous alpha-2-antiplasmin. The invention also relates to a treatment for pulmonary embolism, myocardial infarction, thrombosis or stroke in a patient comprising coadministrating an alpha-2-antiplasmin-binding molecule of the invention together with a thrombolytic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit to U.S. application Ser.No. 60/026,356, filed Sep. 20, 1996, which disclosure is herebyincorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This invention was made in part with Government support underContract #HL-02348 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to a composition and method oftreatment for pulmonary embolism, myocardial infarction, thrombosis, andstroke in a patient, and more specifically to a therapy which enhancesfibrinolysis comprising administering an alpha-2-antiplasmin-bindingmolecule. The invention also relates to a treatment for enhancingfibrinolysis comprising administering an alpha-2-antiplasmin-bindingmolecule together with a thrombolytic agent.

[0005] 2. Description of Background Art

[0006] Venous thrombosis and pulmonary embolism are major causes ofmorbidity and mortality in the United States, accounting for about270,000 hospitalizations a year (Anderson, F. A., Jr. et al., Arch.Intern. Med. 151:933-938 (1991)). In addition, it is estimated thatabout 50,000-200,000 patients a year die from pulmonary embolism(Lilienfeld, D. E. et al., Chest 98:1067-1072 (1990)). In surprisingcontrast with the mortality rate for myocardial infarction, themortality rate for pulmonary embolism (estimated at 9.2% in treatedpatients) has not improved in the last 30 years (Lilienfeld, D. E. etal., Chest 98:1067-1072 (1990); Giuntini, C. et al., Chest 107:3S-9S(1995)). Moreover, survivors of venous thromboembolism are known to beat risk for recurrent thrombosis, postphlebitic syndrome, and pulmonaryhypertension (Sutton, G. C. et al., Br. Heart J. 39:1135-1192 (1977);Salzman, E. W. and Hirsch, J., “The Epidemiology, Pathogenesis andNatural History of Venous Thrombosis,” in Hemostasis and Thrombosis:Basic Principles and Clinical Practice, Coleman, R. W. et al., eds., 3rded. Philadelphia, Pa. (1994), pp. 1275-1296).

[0007] A. Mechanism of Clot Formation and Lysis

[0008] Clots (or thrombi in a patient) are composed of fibrin and bloodplatelets in various ratios. The fundamental reaction in blood clottinginvolves the conversion of a soluble plasma protein (fibrinogen) intoinsoluble fibrin. The conversion of fibrinogen into fibrin is catalyzedby the enzyme, thrombin, which is a serine protease.

[0009] Clot lysis is mediated by plasmin. Under natural conditions,plasminogen is converted to plasmin by plasminogen activators. Naturalplasmin inhibitors include α2-antiplasmin, α2-macroglobulin andα-1-antitrypsin, all glycoproteins. Alpha-2-antiplasmin has a muchhigher affinity for plasmin than α2-macroglobulin and binds specificallyto plasmin in a 1:1 ratio. The larger pool of α-macroglobulin acts as areservoir inhibitor (Kane, K. K., Ann. Clin. Lab. Sci. 14:443-449(1984)). Thus, clot lysis by the administration of t-PA is limited bythe rapid and irreversible inactivation of plasmin by plasmininhibitors.

[0010] B. Treatment for Venous Thrombosis and Pulmonary Embolism

[0011] Standard therapy for venous thromboembolism is heparin, whichpotentiates thrombin and factor Xa inhibition by antithrombin III(Goldhaber, S., Chest 107:45S-51S (1995)). Although heparin decreasesnew thrombus (clot) formation, clinical studies suggest that there islittle early endogenous lysis of the large thrombi that often exist atthe time of diagnosis in patients with venous thromboembolism(Goldhaber, S. Z. et al., Lancet 2:886-889 (1986); “The UrokinasePulmonary Embolism Trial,” Circulation 47:1-108 (1973); Goldhaber, S. Z.et al., Am. J. Med. 88:235-240 (1990); Goldhaber, S. Z. et al., Lancet341:507-511 (1993)). Since large thrombi are associated with an increasein morbidity and mortality, several studies have examined the effects ofplasminogen activators in patients with venous thromboembolism(Goldhaber, S. Z. et al., Lancet 2:886-889 (1986); “The UrokinasePulmonary Embolism Trial,” Circulation 47:1-108 (1973); Goldhaber, S. Z.et al., Am. J. Med. 88:235-240 (1990); Goldhaber, S. Z. et al., Lancet341:507-511 (1993)).

[0012] Compared with heparin alone, plasminogen activators causesignificant increases in the lysis of venous thromboemboli, but patientsare frequently left with large amounts of residual thrombi in the lungsor deep veins immediately after therapy (Goldhaber, S. Z. et al., Lancet2:886-889 (1986); “The Urokinase Pulmonary Embolism Trial,” Circulation47:1-108 (1973); Goldhaber, S. Z. et al., Am. J. Med 88:235-240 (1990);Goldhaber, S. Z. et al., Lancet 341:507-511 (1993)). None of therandomized, controlled trials of patients with pulmonary embolism havedemonstrated a mortality benefit from plasminogen activators, althoughthis may well be due to the small numbers of patients enrolled in thesestudies. Use of plasminogen activators for myocardial infarctions hasshown that 45-70% of patients with coronary thrombosis have failed toachieve full 90 minutes reperfusion with these agents.

[0013] Why venous thromboemboli resist fibrinolysis is unknown. Physicalcharacteristics such as size, retraction, exposure to blood flow, andage may affect the lysis of these large fibrin-rich thrombi (Prewitt, R.M., Chest 99:157S-164S (1991)). However, it is also likely that thefibrinolytic resistance of these thrombi is regulated by specificmolecular factors such as factor XIII, plasminogen activator inhibitor 1(PAI-1), and alpha-2-antiplasmin (α2AP) (Collen, D., Eur. J. Biochem.69:209-216 (1976); Moroi, M. and Aoki, N., J. Biol. Chem. 251:5956-5965(1976); Mullertz, S. and Clemmensen, I., Biochem. J. 159:545-553 (1976);Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982); Robbie, L.A. et al., Thromb. Haemostas. 70:301-306 (1993); Francis, C. W. andMarder, V. J., J. Clin. Invest. 80:1459-1465 (1987); Jansen, J. W. C. M.et al., Thromb. Haemostas. 57:171-175 (1987); Reed, G. L. et al., Trans.Assoc. Am. Phys. 104:21-28 (1991); Stringer, H. A. and Pannekoek, H., J.Biol. Chem. 270:11205-11208 (1995); Carmeliet, P. et al., J. Clin.Invest. 92:2756-2760 (1993); Lang, I. M. et al., Circulation89:2715-2721 (1994); Marsh, J. J. et al., Circulation 90:3091-3097(1994)).

[0014] Because α2AP is an ultrafast covalent inhibitor of plasmin (theenzyme that degrades thrombi), α2AP is a particularly likely cause ofthrombus resistance (Collen, D., Eur. J. Biochem. 69:209-216 (1976);Moroi, M. and Aoki, N., J. Biol. Chem. 251:5956-5965 (1976); Mullertz,S. and Clemmensen, I., Biochem. J. 159:545-553 (1976)). Moreover, a2APis the only fibrinolytic inhibitor that is covalently crosslinked to thefibrin surface (Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542(1982)). This crosslinking (by activated factor XIII) concentrates α2APon the fibrin surface, where it inhibits the initiation of fibrinolysis(Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982)). Previousin vitro studies have shown that clots from α2AP-deficient patients lysespontaneously, suggesting that α2AP plays a critical role in thrombusresistance to endogenous plasminogen activators (Aoki, N. et al., Blood62:1118-1122 (1983); Miles, L. A. et al., Blood 59:1246-1251 (1982)).These observations led to the hypothesis that α2AP is a molecularmediator of the thrombus resistance seen in patients with pulmonaryembolism. To test this hypothesis, we generated a specific inhibitor ofα2AP and used it to determine the role played by α2AP in the regulationof lysis of experimental pulmonary emboli.

[0015] If an individual has formed a fibrin clot (thrombus) prior to theavailability of medical assistance, the clot may be dissolved throughthe use of agents capable of lysing the fibrin thrombus, and therebypermitting blood to again flow through the affected blood vessel. Suchagents include plasmin, anti-coagulants (such as, for example, heparin,hirudin and activated protein C), plasminogen activators (such as, forexample, streptokinase, prourokinase, urokinase, tissue-type plasminogenactivator, staphylokinase, and vampire bat plasminogen activator), andother such agents (Ganz, W. et al., J. Amer. Coll. Cardiol. 1:1247-1253(1983); Rentrop, K. P. et al., Amer. J. Cardiol. 54:29E-31E (1984);Gold, H. K. et al., Amer. J. Cardiol. 53: 122C-125C (1984)).

[0016] At present, treatment of pulmonary embolism, myocardialinfarction, thrombosis, and stroke is partially achieved through theadministration of thrombolytic agents. Use of such agents in therapyoften results in incomplete lysis, and promotes the reformation ofthrombi and reocclusion of the affected blood vessels. Hence, a needexists for an improvement in thrombolytic therapy which enhancesfibrinolysis, while minimizing fibrinogen breakdown and preventingreformation of thrombi.

[0017] C Alpha-2 Antiplasmin Antibodies

[0018] Alpha-2-antiplasmin (α2AP) has three functional domains: thereactive site for plasmin, the plasmin(ogen) or LBS-binding site[complementary to the LBS (lysine-binding site) of plasmin(ogen)], andthe crosslinking site for fibrin. Mimuro, J. et al., Blood 69:446-453(1987). Mimuro et al. discloses antibodies to α2AP, one of which(JPTI-1) was specific to the reactive site of α2AP and preventedformation of α2AP complexes, thereby inhibiting antiplasmin activity.However, Mimuro et al. does not teach administration of the JPTI-1antibody to enhance clot lysis. Other antibodies specific for α2AP aretaught by Plow, E. F. et al., J. Biol. Chem. 255:2902-2906 (1980);Wimen, B. et al., Scan. J. Clin. Lab. Invest. 43:27-33 (1983); Hattey,E. et al., Thromb. Res. 45:485-495 (1987); Collen, U.S. Pat. No.4,346,029 (1980); and Collen, U.S. Pat. No. 4,198,335 (1980).

SUMMARY OF THE INVENTION

[0019] The present invention relates to an improved thrombolytic therapyfor the treatment of pulmonary embolism, myocardial infarction,thrombosis and stroke in patients. The invention is directed to animmunologic molecule capable of binding to both (1) human and nonhumancirculating α2-antiplasmins and (2) human and nonhuman fibrincrosslinked α2-antiplasmins. In preferred embodiments, the immunologicmolecule is a chimeric antibody, a humanized antibody, or a single chainantibody. The invention is also directed to a method for treatingpulmonary embolism, myocardial infarction, thrombosis and stroke in apatient comprising administering an α2-antiplasmin-binding moleculecapable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins. The invention further provides a method of treatmentfor pulmonary embolism, myocardial infarction, thrombosis and stroke ina patient which comprises co-administrating to a patient in need of suchtreatment:

[0020] (a) a therapeutically effective amount of an immunologic moleculecapable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins; and

[0021] (b) a therapeutically effective amount of a thrombolytic agent,wherein the immunologic molecule (a) is different from the thrombolyticagent (b), thereby treating the patient.

[0022] The invention provides a monoclonal antibody or fragment thereofwherein the monoclonal antibody is capable of binding to both (1) humanand nonhuman circulating α2-antiplasmins and (2) human and nonhumanfibrin crosslinked α2-antiplasmins. In one embodiment, the invention ismonoclonal antibody 77A3. In another embodiment, the invention ismonoclonal antibody 49C9. In another embodiment, the monoclonal antibodyis 70B 11.

[0023] The invention also provides a method of making the monoclonalantibody comprising:

[0024] (a) immunizing an animal with α2-antiplasmin or fragment thereof;

[0025] (b) fusing cells from the animal with tumor cells to make ahybridoma cell line;

[0026] (c) cloning the hybridoma cell line;

[0027] (d) selecting for the monoclonal antibody capable of binding toboth (1) human and nonhuman circulating α2-antiplasmins and (2) humanand nonhuman fibrin crosslinked α2-antiplasmins; and

[0028] (e) obtaining the monoclonal antibody.

[0029] The invention provides a hybridoma cell line which produces themonoclonal antibody capable of binding to both (1) human and nonhumancirculating α2-antiplasmins and (2) human and nonhuman fibrincrosslinked α2-antiplasmins. In one embodiment, the invention ishybridoma cell line 77A3 (ATCC Accession No. HB-12192; Deposited at theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,20862 on Sep. 20, 1996).

[0030] The invention is directed to a method of making the hybridomacell line comprising:

[0031] (a) immunizing an animal with α2-antiplasmin or fragment thereof;

[0032] (b) fusing the cells from the animal with tumor cells to make thehybridoma cell line; and

[0033] (c) obtaining the hybridoma cell line which produces themonoclonal antibody capable of binding to both (1) human and nonhumancirculating α2-antiplasmins and (2) human and nonhuman fibrincrosslinked α2-antiplasmins.

[0034] The invention also provides a method for treating a number ofdiseases and conditions, including pulmonary embolism, myocardialinfarction, thrombosis and stroke in a patient comprising administeringa therapeutically effective amount of an immunologic molecule which iscapable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins, thereby treating the patient.

[0035] The invention further provides a method of treatment forpulmonary embolism, myocardial infarction, thrombosis or stroke in apatient which comprises co-administering to a patient in need of suchtreatment:

[0036] (a) a therapeutically effective amount of an immunologic moleculewhich is capable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins; and

[0037] (b) a therapeutically effective amount of a thrombolytic agent,wherein the immunologic molecule (a) is different from the thrombolyticagent (b), thereby treating the patient.

[0038] In preferred embodiments, the thrombolytic agent is plasmin,anti-coagulant, or plasminogen activator. In one embodiment, theanti-coagulant is selected from the group consisting of heparin, hirudinand activated protein C. In another embodiment, the plasminogenactivator is selected from the group consisting of staphylokinase,streptokinase, prourokinase, urokinase, tissue-type plasminogenactivator, and vampire bat plasminogen activator.

[0039] Other embodiments of the invention include, the immunologicmolecule provided to the patient by an intravenous infusion, by anintravenously injected bolus, or with a first bolus containing theimmunologic molecule (a) and a subsequently administered second boluscontaining the thrombolytic agent (b). Further embodiments include, theimmunologic molecule (a) provided to the patient at a dose of between 3to 300 nmole per kg of patient weight; and the thrombolytic agent (b)provided to the patient at a dose of between 0.01 to 3.0 mg per kg ofpatient weight.

[0040] The invention provides a kit useful for carrying out the methodof treatment for pulmonary embolism, myocardial infarction, thrombosisor stroke in a patient, being compartmentalized in close confinement toreceive two or more container means therein, which comprises:

[0041] (1) a first container containing a therapeutically effectiveamount of the immunologic molecule (a); and

[0042] (2) a second container containing a therapeutically effectiveamount of the thrombolytic agent (b), wherein the immunologic molecule(a) is different from the thrombolytic agent (b).

[0043] The invention also provides nucleic acid molecules encodingimmunologic molecules capable of binding to both (1) human and nonhumancirculating α2-antiplasmins and (2) human and nonhuman fibrincrosslinked α2-antiplasmins. Also provided are molecules comprising anamino acid sequence of the binding region of an immunologic moleculedescribed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1. Comparison of binding to ¹²⁵I-α2-antiplasmin of monoclonalantibodies 49C9, 70B11, 77A3, RWR and anti-digoxin (control). Wells of amicrotiter plate were coated with goat antimouse antibody. The wellswere incubated in duplicate with 49C9, 70B11, 77A3, RWR or a control(antidigoxin) MAb (Mudgett-Hunter, M. et al., Mol. Immunol. 22:477-488(1985)). After a wash, ¹²⁵I-α2AP (60,000 cpm) was added for an hour. Thewells were rinsed and the amount of bound ¹²⁵I-α2AP was measured in agamma counter.

[0045]FIG. 2. Competition binding assays of monoclonal antibodies 49C9,70B11, 77A3, RWR and anti-digoxin with immobilized 70B11. Competitionradioimmunoassay were performed by coating wells of a microtiter platewith 25 μl of purified MAb (70B11) in duplicate (10 μg/ml) for 1 hour.The wells were washed and blocked with 1% BSA for 1 hour. After washing,25 μl of a competitor MAb, same MAb or negative control MAb was added todifferent wells (50 μg/ml) followed by 25 μl of ¹²⁵I-α2-antiplasmin(100,000 cpm). After 1 hour incubation, the wells were washed, cut andthe radioactivity was measured in a gamma scintillation counter.

[0046]FIG. 3. Comparison of amount of lysis by different monoclonalantibodies (or TBS alone) as a function of dose of urokinase. SeeExample 1, below, for detailed description of the method. The amount oflysis was determined by gamma counting. The percent lysis was defined at100×(total supernatant cpm÷total clot cpm).

[0047]FIG. 4. Dose response studies in the absence or presence of MAb77A3. Lysis by urokinase is increased approximately 100-fold by 77A3.

[0048]FIG. 5. Reduced SDS-polyacrylamide gel electrophoresis of 77A3purification. Ascites containing 77A3 were harvested and purified. Lane1, protein standards with molecular mass in kDa (left); lane 2,supernatant after precipitation with 40% ammonium sulfate; lane 3,purified 77A3. The reduced 77A3 immunoglobulin consists of bands of ˜50kDa, corresponding to the heavy chain, and ˜25 kDa, corresponding to thelight chain.

[0049]FIG. 6. Effect of 77A3 on the rate of lysis of ferret plasma clotsin vitro. Ferret plasma clots formed with trace amounts of ¹²⁵I-labeledhuman fibrinogen were incubated with 100 μl of TBS (control) or purifiedMAb (25 μg, 77A3 or RWR). Clot lysis was initiated by adding 0.1 unit ofrt-PA per tube. The clots were incubated at 37° C. and the amount oflysis was determined by sampling for the release of radiolabeled fibrindegradation products into the supernatant as described (Reed, G. L. IIIet al., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990)).

[0050]FIG. 7. Effect of in vivo administration of MAb 77A3 on functionalα2AP levels in ferrets. In dose finding experiments, two anesthetizedferrets (A, B) were given 77A3 intravenously (22.5 mg/kg) and the amountof functional α2AP was measured in citrated plasma samples drawn before(time 0) and 1 and 4 hours after infusion. The data represent themean±SD inhibition of α2AP in plasma samples.

[0051]FIG. 8. Effect of rt-PA and α2AP inhibition on the lysis ofpulmonary emboli in vivo. Anesthetized ferrets were given a heparinbolus (100 U/kg) and ¹²⁵I-labeled fibrin clots were embolized into thelungs. After embolization, three groups of ferrets were given rt-PA (0,1, or 2 mg/kg) over 2 hours intravenously (plain bars). Two other groupsof ferrets also received rt-PA (1 mg/kg) and a control MAb (antidigoxin,black bar, 22.5 mg/kg) or a MAb that inhibits α2AP (77A3, striped bar,same dose). The graph shows the amount of lysis (mean±SD) for eachtreatment group. The number of ferrets in each treatment group is shown,and the P values for differences between groups are indicated.

[0052]FIG. 9. Residual fibrinogen levels in animals treated withheparin, rt-PA, and an α2AP inhibitor. Blood samples were collected (onEDTA with aprotinin) from ferrets before pulmonary embolization and atthe end of the experiment. Residual fibrinogen levels were measured asdescribed (Rampling, M. W. and Gaffney, P. J., Clin. Chim. Acta.67:43-52(1976)). The graph shows the mean±SD percentage residual fibrinogenlevel for animals receiving rt-PA alone (0, 1, or 2 mg/kg; plain bars)and those receiving rt-PA and the α2AP inhibitor (striped bar).

[0053]FIG. 10. The peptide sequences of the amino terminus of purifiedlight chains from 49C9 (SEQ ID NO: 1), 70B11 (SEQ ID NO: 2) and 77A3(SEQ ID NO: 3) are shown.

[0054]FIG. 11. The cDNA sequence (SEQ ID NO: 4) and correspondingdeduced amino acid sequence of the signal peptide (amino acids −20 to −1of SEQ ID NO: 5) and light chain variable regions (amino acids 1 to 107of SEQ ID NO: 5) of 49C9 are shown.

[0055]FIG. 12. The cDNA sequence (SEQ ID NO: 6) and correspondingdeduced amino acid sequence of the signal peptide (amino acids −20 to −1of SEQ ID NO: 7) and light chain variable regions (amino acids 1 to 107of SEQ ID NO: 7) of 70B11 are shown.

[0056]FIG. 13. The cDNA sequence (SEQ ID NO: 8) and correspondingdeduced amino acid sequence of the signal peptide (amino acids −20 to −1of SEQ ID NO: 9) and light chain variable regions (amino acids 1 to 107of SEQ ID NO: 9) of 77A3 are shown.

[0057]FIG. 14. The cDNA sequence (SEQ ID NO: 10) and correspondingdeduced amino acid sequence of the signal peptide (amino acids −19 to −1of SEQ ID NO: 11) and heavy chain variable regions (amino acids 1-119 ofSEQ ID NO: 11) of 49C9 are shown.

[0058]FIG. 15. The cDNA sequence (SEQ ID NO: 12) and correspondingdeduced amino acid sequence of the signal peptide (amino acids −19 to −1of SEQ ID NO: 13) and heavy chain variable regions (amino acids 1-119 ofSEQ ID NO: 13) of70B11 are shown.

[0059]FIG. 16. The cDNA sequence (SEQ ID NO: 14) and correspondingdeduced amino acid sequence of the signal peptide (amino acids −19 to −1of SEQ ID NO: 15) and heavy chain variable regions (amino acids 1-119 ofSEQ ID NO: 15) of 77A3 are shown.

[0060]FIG. 17. The cDNA sequence (SEQ ID NO: 16) and corresponding aminoacid sequence (SEQ ID NO: 17) of humanized77A3-1 and humanized 77A3-2light chain. Positions falling within the CDR loops are shown enclosedwithin the boxes with solid borders.

[0061]FIG. 18. The cDNA sequence (SEQ ID NO: 18) and corresponding aminoacid sequence (SEQ ID NO: 19) of humanized 77A3-1 heavy chain. Positionsfalling within the CDR loops are shown enclosed within the boxes withsolid borders.

[0062]FIG. 19. The cDNA sequence (SEQ ID NO: 20) and corresponding aminoacid sequence (SEQ ID NO: 21) of humanized 77A3-2 heavy chain. Positionsfalling within the CDR loops are shown enclosed within the boxes withsolid borders.

[0063]FIG. 20. Results of murine 77A3 (X), chimeric 77A3 () andhumanized 77A3-1 (▪) in the plasmin assay with chromogenic substrate areshown.

[0064]FIG. 21. The amino acid sequences of the light chains are shown:h77A3-1 and h77A3-2 (SEQ ID NO: 17); m77A3 (SEQ ID NO: 9); m49C9 (SEQ IDNO: 5); m70B11 (SEQ ID NO: 7); murine consensus (SEQ ID NO: 75), whichshows the consensus between m77A3, m49C9, and m70B11; 77A3/49C9consensus (SEQ ID NO: 76), which shows the consensus between 77A3 and49C9; and all (SEQ ID NO: 77), which shows the consensus betweenh77A3-1, h77A3-2, m77A3, m49C9, and m70B11. Positions falling withingthe CDR loops are shown enclosed within the boxes.

[0065]FIG. 22. The amino acid sequences of the heavy chains are shown.h77A3-1 (SEQ ID NO: 19); h77A3-2 (SEQ ID NO: 21); m77A3 (SEQ ID NO: 15);m49C9 (SEQ ID NO: 11); m70B11 (SEQ ID NO: 13); humanized consensus (SEQID NO: 78), which is the consensus between h77A3-1 and h77A3-2; murineconsensus (SEQ ID NO: 79), which is the consensus between m77A3, m49C9,and m70B11; 77A3/49C9 consensus (SEQ ID NO: 80), which is the consensusbetween 77A3 and 49C9; and all (SEQ ID NO: 81), which is the consensusbetween h77A3-1, h77A3-2, m77A3, m49C9, and m70B11. Positions fallingwithing the CDR loops are shown enclosed within the boxes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Alpha-2-antiplasmin (α2AP) is a molecular mediator of thethrombus resistance in patients with pulmonary embolism. A specificinhibitor of α2AP is described which is used to determine the roleplayed by α2AP in the regulation of fibrinolysis.

[0067] A. Immunologic Molecules

[0068] In the following description, reference will be made to variousmethodologies well-known to those skilled in the art of immunology.Standard reference works setting forth the general principles ofimmunology include Klein, J., Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, New York (1982); Kennett, R. et al.,Monoclonal Antibodies, Hybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, New York (1980); Campbell, A., “MonoclonalAntibody Technology,” in Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Burdon, R., et al., eds., Elsevier,Amsterdam (1984); and Eisen, H. N., Microbiology, 3rd ed, Davis, B. D.,et al., Harper & Row, Philadelphia (1980).

[0069] As used herein, α2AP-binding molecule includes antibodies(polyclonal or monoclonal), as well as ligands. As used herein, an“immunologic molecule” refers to polypeptides comprising the bindingregion of a monoclonal antibody. Thus, monoclonal antibodies, antibodyfragments, chimeric antibodies, humanized antibodies, and fusionproteins comprising antibody binding regions are “immunologicmolecules”. The term “antibody” (Ab) or “monoclonal antibody” (MAb) ismeant to include intact molecules as well as antibody fragments (suchas, for example, Fv, Fab and F(ab′)₂ fragments), single chainantigen-binding proteins, “humanized” antibodies, and chimericantibodies which are capable of specifically binding to α2AP. Fab andF(ab′)₂ fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). Thus, these fragments are preferred.

[0070] An antibody is said to be “capable of binding” a molecule if itis capable of specifically reacting with the molecule to thereby bindthe molecule to the antibody. As used herein, the term “hapten” isintended to refer to any molecule capable of being bound by an antibody.The term “epitope” is meant to refer to that portion of a hapten whichcan be recognized and bound by an antibody. A hapten or antigen may haveone, or more than one epitope. An “antigen” or “immunogen” is a haptenwhich is additionally capable of inducing an animal to produce antibodycapable of binding to an epitope of that antigen. The specific reactionreferred to above is meant to indicate that the hapten will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

[0071] The antibodies of the present invention may be prepared by any ofa variety of methods. For example, cells expressing α2AP (or fractions,lysates, etc. thereof) can be administered to an animal in order toinduce the production of sera containing polyclonal antibodies that arecapable of binding α2AP. In a preferred method, a preparation of α2AP ofthe present invention is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

[0072] The antibodies of the present invention may also be preparedusing phage display technology. Methods of preparing antibodies usingphage display are known in the art. See, for example, U.S. Pat. No.5,565,332; Clarkson et al., Nature 352:624-628 (1991); Huse, Science246:1275-1281 (1989); Kang, Proc. Natl. Acad Sci. USA 88:11120-11123(1993); Marks, J. Mol. Biol. 222:581-597 (1991); and McCafferty et al.,Nature 348:552-554 (1990).

[0073] In one preferred method, the immunogenic molecules of the presentinvention are monoclonal antibodies (or α2AP binding molecules). Suchmonoclonal antibodies can be prepared using hybridoma technology (Kohleret al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al.,in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.563-681 (1981)). In general, such procedures involve immunizing ananimal (preferably a mouse) with the antigen or with a cell whichexpresses the antigen. A preferred antigen is purified α2AP. The mostpreferred antigen is α2AP fragment (fibrin binding region) obtained bytrypsin digest of a plasma clot, then affinity purified with aSEPHAROSE-coupled monoclonal antibody, RWR (Reed, G. L. III et al.,Trans. Assoc. Am. Phys. 101:250-256 (1988); U.S. Pat. No. 5,372,812,issued Dec. 13, 1994). Suitable cells can be recognized by theircapacity to secrete anti-α2AP antibody. Such cells may be cultured inany suitable tissue culture medium; however, it is preferable to culturecells in Earle's modified Eagle's medium supplemented with 10% fetalbovine serum (inactivated at about 56° C.), and supplemented with about10 μg/l of nonessential amino acids, about 1,000 U/ml of penicillin, andabout 100 μg/ml of streptomycin. The splenocytes of such mice areextracted and fused with a suitable myeloma cell line. The method ofsomatic cell fusion is described in Galfre, G. and Milstein, C., Meth.Enzymol. 73:3-46 (1981). After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al., Gastroenterology 80:225-232(1981). The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of bindingα2AP.

[0074] Alternatively, additional antibodies capable of binding to theα2AP antigen may be produced in a two-step procedure through the use ofanti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, α2AP-specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theα2AP-specific antibody can be blocked by the α2AP antigen. Suchantibodies comprise anti-idiotypic antibodies to the α2AP-specificantibody and can be used to immunize an animal to induce formation offurther α2AP-specific antibodies.

[0075] It will be appreciated that Fab and F(ab′)₂ and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). Alternatively,α2AP-binding fragments can be produced through the application ofrecombinant DNA technology, through synthetic chemistry, orbiotinylation.

[0076] Also intended within the scope of the present invention arehumanized or chimeric antibodies, produced using genetic constructsderived from hybridoma cells producing the MAbs described above.Humanized antibodies are antibodies in which the framework or otherregions of the murine Ab is replaced with the homologous regions of anonmurine antibody. Chimeric antibodies are antibodies in which themurine constant region has been replaced with a non-murine constantregion. Methods for production of chimeric antibodies are known in theart. See, for review: Morrison, Science, 229:1202-1207 (1985); Oi etal., BioTechniques 4:214 (1986); see also, Cabilly et al., U.S. Pat. No.4,816,567 (Mar. 28, 1989); Taniguchi et al., EP171496 (Feb 19, 1986);Morrison et al., EP173494 (Mar. 5, 1986); Neuberger et al., WO8601533(Mar. 13, 1986); Robinson et al., WO 8702671 (May 7, 1987); Boulianne etal., Nature 312:643-646 (1984); and Neuberger et al., Nature 314:268-270(1985). Methods for production of humanized antibodies are known in theart. See, for example, U.S. Pat. No. 5,585,089; Jones et al., Nature321:522-525 (1986); and Kettleborough et al., Protein Engineering4:773-783 (1991).

[0077] Also provided in the present invention are single-chainantibodies capable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins. Methods of making single chain antibodies are wellknown in the art. See, for example, U.S. Pat. No. 4,946,778; U.S. Pat.No. 5,260,203; U.S. Pat. No. 5,091,513; and U.S. Pat. No. 5,455,030, allof which are herein incorporated by reference.

[0078] Also intended within the scope of the present invention arevariants of the monoclonal antibodies described above.

[0079] The present inventors have determined the nucleotide and aminoacid sequence of several immunologic molecules capable of binding toboth (1) human and nonhuman circulating α2-antiplasmins and (2) humanand nonhuman fibrin crosslinked α2-antiplasmins. Accordingly, thepresent invention provides for nucleic acid molecules comprising anucleotide sequence encoding for an immunologic molecule of the presentinvention or fragment thereof Due to the degeneracy of the genetic code,and to the fact that the genetic code is known, all other nucleotidesequences which encode the same amino acid sequence as the nucleotidesof the present invention can be determined and used in the practice ofthe present invention.

[0080] DNA clones containing nucleotide sequences encoding the followingantibody chains were deposited at the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md., 20862 on Sep. 19, 1997: lightchain of 77A3 (77A3 LC), ATCC Accession No. ______ ; light chain of 49C9(49C9 LC), ATCC Accession No. ______ ; light chain of 70B11 (70B11 LC),ATCC Accession No. ______ ; heavy chain of 77A3 (77A3 HC), ATCCAccession No. ______ ; heavy chain of 49C9 (49C9 HC), ATCC Accession No.______ ; and heavy chain of 70B11 (70B11 HC), ATCC Accession No. ______.

[0081] The nucleic acid molecules of the present invention include:nucleic acid molecules containing a nucleotide sequence encoding themature light chain of 77A3 as shown in SEQ ID NO: 9 or as encoded by theclone contained in the ATCC Accession No. ______ ; nucleic acidmolecules containing a nucleotide sequence encoding the mature lightchain of 49C9 as shown in SEQ ID NO: 5 or as encoded by the clonecontained in ATCC Accession No. ______ ; and nucleic acid moleculescontaining a nucleotide sequence encoding the mature light chain of70B11 as shown in SEQ ID NO: 7 or as encoded by the clone contained inATCC Accession No. ______ .

[0082] Also included in the present invention are nucleic acid moleculescontaining a nucleotide sequence encoding an antibody heavy chain,including: nucleic acid molecules containing a nucleotide sequenceencoding the mature heavy chain of 77A3 as shown in SEQ ID NO: 15 or asencoded by the clone contained in ATCC Accession No. ______ ; nucleicacid molecules containing a nucleotide sequence encoding the matureheavy chain of 49C9 as shown in SEQ ID NO: 11 or as encoded by the clonecontained in ATCC Accession No. ______ ; and nucleic acid moleculescontaining a nucleotide sequence encoding the mature heavy chain of70B11 as shown in SEQ ID NO: 13 or as encoded by the clone contained inATCC Accession No. ______ .

[0083] Also included are nucleic acid molecules encoding humanizedantibodies including: nucleic acid molecules comprising a nucleotidesequence encoding for amino acid residues 1 to 107 of SEQ ID NO: 17;nucleic acid molecules comprising a nucleotide sequence encoding foramino acid residues 1 to 119 of SEQ ID NO: 19; and nucleic acidmolecules comprising a nucleotide sequence encoding for amino acidresidues 1 to 119 of SEQ ID NO: 21.

[0084] Also intended within the scope of the invention are nucleic acidmolecules encoding “consensus” amino acid sequences of heavy and lightchain of antibodies, including: nucleic acid molecules comprising anucleotide sequence encoding for amino acid residues 1 to 107 of SEQ IDNO: 75; nucleic acid molecules comprising a nucleotide sequence encodingfor amino acid residues 1 to 107 to of SEQ ID NO: 76; nucleic acidmolecules comprising a nucleotide sequence encoding for amino acidresidues 1 to 107 of SEQ ID NO: 77; nucleic acid molecules comprising anucleotide sequence encoding for amino acid residues 1 to 119 of SEQ IDNO: 78; nucleic acid molecules comprising a nucleotide sequence encodingfor amino acid residues 1 to 119 of SEQ ID NO: 79; nucleic acidmolecules comprising a nucleotide sequence encoding for amino acidresidues 1 to 119 of SEQ ID NO: 80; and nucleic acid moleculescomprising a nucleotide sequence encoding for amino acid residues 1 to119 of SEQ ID NO: 81.

[0085] Nucleic acid molecules encoding an immunologic molecule of thepresent invention can be used to express recombinant proteins. A nucleicacid molecule encoding an immunologic molecule of the present inventioncan be inserted into a vector in accordance with conventionaltechniques. A “vector” should be understood as a nucleic acid vehicleused for cloning or expressing a desired sequence in a host.

[0086] In one embodiment, the recombinant vector is capable ofexpressing the immunologic molecule of the present invention. A vectoris said to be “capable of expressing” a polypeptide if it contains anucleotide sequence that encodes for the polypeptide, as well astranscriptional and translational regulator information operably linkedto the nucleotide sequence that encodes the polypeptide. Two nucleotidesequences are said to be “operably linked” if the nature of the linkagebetween the two nucleotide sequences does not: result in theintroduction of a frame-shift mutation; interfere with the ability ofthe promoter region sequence to direct the transcription of the desiredsequence; or interfere with the ability of the desired sequence to betranscribed by the promoter region sequence. Thus, a promoter regionwould be operably linked to a desired nucleotide sequence if thepromoter were capable of effecting transcription of that nucleotidesequence.

[0087] Once the recombinant vector is constructed, it can be introducedinto a host cell, either prokaryotic or eukaryotic, by a variety ofconventional techniques including transfection, transduction,electroporation, calcium-phosphate precipitation, and microinjection.Prokaryotic hosts include bacteria such as E. coli, Bacillus,Streptomyces, and Salmonella. The most preferred prokaryotic host is E.coli. Eukaryotic hosts include yeast cells, insect cells, and mammaliancells, such as COS cells, CHO cells, and myeloma cells. In oneembodiment of the invention, CHO cells are preferred.

[0088] In one embodiment of the invention, a nucleic acid moleculecomprising a nucleotide sequence encoding for the light chain of anantibody is introduced into a vector, and a nucleic acid moleculecomprising a nucleotide sequence encoding for the heavy chain of anantibody is introducing into another vector. Both vectors are introducedinto the same host cell. Alternatively, both chains could be introducedinto the same vector.

[0089] Following expression in an appropriate host, the polypeptide canbe readily isolated using standard techniques, including affinitychromatography.

[0090] Also intended within the scope of the present invention aremolecules comprising an amino acid sequence of the binding region of animmunologic molecule described herein. Molecules comprising an aminoacid sequence of the binding region of an immunologic molecule describedherein include, but are not limited to, monoclonal antibodies, humanizedantibodies, chimeric antibodies, fragments of any such antibodies,single chain antibodies, fusion proteins, and the like. Such moleculescan be used in the assays and methods of treatment of the presentinvention described below.

[0091] The amino acid sequence of the binding region of the immunologicmolecules of the present invention are shown in FIG. 21 for the lightchains and FIG. 22 for the heavy chains. In FIG. 21, the amino acidsequence of the binding regions of the light chains of h77A3-1 andh77A3-2 (amino acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ IDNO: 17), m77A3 (amino acid residues 24 to 34, 50 to 56 and 89 to 97 ofSEQ ID NO: 9), m44C9 (amino acid residues 24 to 34, 50 to 56 and 89 to97 of SEQ ID NO: 5), m70B11 (amino acid residues 24 to 34, 50 to 56 and89 to 97 of SEQ ID NO: 7), the murine consensus (amino acid residues 24to 34, 50 to 56 and 89 to 97 of SEQ ID NO: 75), the 77A3/49C9 consensus(amino acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ ID NO: 76)and the consensus of all light chains (amino acid residues 24 to 34, 50to 56 and 89 to 97 of SEQ ID NO: 77) are shown in the larger boxes.

[0092] In FIG. 22, the amino acid sequence of the binding regions of theheavy chains of h77A3-1 (amino acid residues 26 to 35, 50 to 66 and 99to 108 of SEQ ID NO: 19), h77A3-2 (amino acid residues 26 to 35, 50 to66 and 99 to 108 of SEQ ID NO: 21), m77A3 (amino acid residues 26 to 35,50 to 66 and 99 to 108 of SEQ ID NO: 15), m49C9 (amino acid residues 26to 35, 50 to 66 and 99 to 108 of SEQ ID NO: 11), m70B11 (amino acidresidues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO: 13), thehumanized consensus (amino acid residues 26 to 35, 50 to 66 and 99 to108 of SEQ ID NO: 78), the murine consensus (amino acid residues 26 to35, 50 to 66 and 99 to 108 of SEQ ID NO: 79), the 77A3/49C9 consensus(amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO: 80),and the consensus of all the heavy chains (amino acid residues 26 to 35,50 to 66 and 99 to 108 of SEQ ID NO: 81) are shown in the overlappingboxes.

[0093] B. Assays

[0094] Methods for immunoblotting are known in the art (see, forexample, Reed, G. L. et al., J. Immunol. 150:4407-4415 (1993)). In apreferred method, the α2AP is electrophoresed on a slab minigel underreducing and non-reducing conditions. The gel is electroblotted topolyvinylidene difluoride membrane. The blot is exposed to differenthybridoma supernatants in the channels of a miniblotter apparatus. Afterwashing, the bound antibody is detected by incubation with ¹²⁵I-goatantimouse antibody. After additional washing, the membrane is exposed ina phosphorimager (Molecular Devices, Sunnyvale, Calif.).

[0095] Methods for radioimmunoassays are also known. For example, thewells of a microtiter plate are coated with goat antimouse antibody. Thewells are washed and blocked with BSA. The hybridoma supernatants areadded to the empty wells. After incubation, the wells are washed and¹²⁵I-α2AP is added. After washing, the wells are cut and the boundantibody is measured by gamma scintillation counting. For competitionassays, the wells of a microtiter plate are coated with a competing MAb.In a preferred embodiment, the binding of MAbs to ¹²⁵I-α2AP (preferably,the fibrin binding region fragment of α2AP, obtained by binding to RWR)is assayed by reverse solid-phase radioimmunoassay.

[0096] Methods for clot assays are also known (see, for example, Reed,G. L. III et al., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990). In apreferred embodiment, plasma is mixed with ¹²⁵I-fibrinogen, then clottedby mixing with CaCl₂ and thrombin. Clots are compressed and washed withTris-buffered saline to remove unbound proteins. The supernatant isremoved and the clots counted in a gamma counter. To each set ofduplicate clots is added, various amounts of plasminogen activator,anti-coagulant, and Tris-buffered saline containing the MAb or no MAb.The clots are incubated and at various intervals, a portion of thesolution is temporarily removed and gamma-counted to determine theamount of lysis. The percent lysis may be defined at 100× (totalsupernatant cpm/total clot cpm).

[0097] Fibrinogen assays are known. Blood samples and platelet-poorplasma are assayed for fibrinogen by, for example, the sodium sulfitemethod (Rampling, M. W. and Gaffney, P. J., Clin. Chim. Acta. 67:43-52(1976)).

[0098] Alpha-2-antiplasmin levels in plasma are assayed, for example,with a chromogenic substrate assay for plasmin inhibition (Stachrom kit)as described in Reed, G. L., III et al., Proc. Natl. Acad. Sci. USA87:1114-1118 (1990).

[0099] Statistical tests may be analyzed by, for example, a one wayanalysis of variance followed by a Bonferroni-Dunn procedure formultiple comparison testing.

[0100] In vivo pulmonary embolism experiments are described in Example2, below.

[0101] C. Methods of Treatment

[0102] By “patient” is intended, human or nonhuman. Nonhumans include,for example, baboon, green monkey, dog, cynamologus, marmoset, ferret,guinea pig, and gerbil.

[0103] By “clot” is intended, an in vitro blood or fibrin clot, or“thrombi” in a patient. Diseases treated according to the methods of hisinvention include, but are not limited to pulmonary thromboembolism;acute coronary syndrome, including unstable angina pectoris andnon-Q-wave myocardial infarction; various forms of thrombosis, includingvenous thrombosis (e.g., deep venous thrombosis), and arterialthrombosis (e.g., renal, mesenteric, and limb thrombosis); and cerebraland thrombosis embolism; renal vein and peripheral arterial thrombosis,myocardial infarction, stroke, and other thromboses. This method mayalso be used to treat thrombotic conditions secondary or concomitant tosurgical procedures, including percutaneous transluminal coronaryangioplasty, peripheral arterial angioplasty, bypass graft, and stent.The “treating” or “treatment” is by, for example, inhibiting theformation of a thrombus, dissolving a thrombus, or by enhancingfibrinolysis.

[0104] By the term “co-administration” is intended that each of thehapten-binding molecule and thrombolytic agent will be administeredduring a time frame wherein the respective periods of pharmacologicalactivity overlap. The two agents may be administered simultaneously orsequentially.

[0105] The α2AP-binding molecules of the present invention may bemonoclonal antibodies or fragments thereof It is preferable to employthe F(ab′)₂ fragment of such an antibody for this purpose, in order tominimize any immunological reaction caused by the Fc portion of theimmunoglobulin. Also preferred are single-chain antibodies, such as sFv.Procedures for preparing monoclonal antibodies are disclosed byKaprowski, H. et al., U.S. Pat. No. 4,172,124, and Kohler et al., Nature256:495-497 (1975). The preparation of monoclonal antibodies capable ofpreventing the inhibition of plasmin are taught by Mimuro, J. et al.,Blood 69:446-453 (1987), and described in the examples section of thepresent application.

[0106] As used herein, an “antigen” is a molecule capable of being boundby an antibody such as, for example, α2AP. In order to be used inaccordance with the present invention, the “antigen-binding molecule”must be capable of binding to a plasmin inhibitor and thereby preventsuch an inhibitor from forming inhibitor-plasmin complexes. Any suchantigen-binding molecule may be employed in accordance with the presentinvention. A preferred embodiment is α2AP-binding molecule which iscapable of binding to α2AP or fragment thereof. An especially preferredα2AP-binding molecule for this purpose is a monoclonal antibody.Preferred embodiments of the monoclonal antibody is 77A3, 70B11 or 49C9,described more fully below. The hybridoma producing MAb 77A3 has beendeposited under the terms of the Budapest Treaty with the InternationalDepository Authority American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A., on Sep. 20, 1996; the ATCCAccession No. is HB-12192.

[0107] Also preferred are chimeric an humanized antibodies. Anespecially preferred chimeric antibody for this purpose is c77A3,described more fully below. Especially preferred humanized antibodiesfor this purpose are h77A3-1 and h77A3-2, described more fully below.Also preferred are antibody fragments and single-chain antibodies,including sFv77A3-1 and sFv77A3-2, described below.

[0108] The terms “thrombolytic agent” are meant to refer to any agentcapable of either dissolving a fibrin and/or platelet clot (orthrombus), or inhibiting the formation of such a clot. Examples ofthrombolytic agents include fibrinolytic molecules, such as plasmin,plasminogen activator (for example, staphylokinase, streptokinase,prourokinase, urokinase, tissue-type plasminogen activator, and vampirebat plasminogen activator); anti-coagulants (for example, inhibitors offibrin formation, such as heparin, hirudin and activated protein C; andanti-platelet agents, such as ticlopidine, aspirin, and clopidigrel andinhibitors of glycoprotein IIb/IIIa function). Use of t-PA for thesepurposes is especially preferred. Although natural t-PA may be employed,it is preferable to employ recombinant t-PA (rt-PA). The invention mayadditionally employ hybrids, physiologically active fragments or mutantforms of the above thrombolytic agents. For example, the term“tissue-type plasminogen activator” as used herein is intended toinclude such hybrids, fragments and mutants, as well as both naturallyderived and recombinantly derived tissue-type plasminogen activator.

[0109] As stated, the methods of the invention comprise theadministration of the α2AP-binding molecule alone or in combination witha thrombolytic agent. When administered alone the molecule enhancesendogenous fibrinolysis or thrombolysis by significantly augmenting clotlysis by endogenous plasminogen activators. Further, administration ofthe α2AP-binding molecule does not increase fibrinogen consumption overthat obtained with equivalent doses of t-PA alone. Thus, the presentmethod of clot-specific inhibition of α2AP enhances the potency of theplasminogen activator and preserves its fibrin selectivity.

[0110] Alternatively, the α2AP-binding molecule is administered with athrombolytic agent. In this embodiment, the α2AP-binding molecule andthe thrombolytic agent of the present invention are intended to beco-administered to the recipient. It is preferable to provide theα2AP-binding molecule to the patient prior to the administration of thethrombolytic agent.

[0111] The α2AP-binding molecule of the present invention is providedfor the purpose of preventing the inhibition of plasmin by a plasmininhibitor. It has been discovered that coadministration of theα2AP-binding molecule together with a thrombolytic agent causes asynergistic effect, and thereby enhances clot lysis (thrombolysis) to agreater extent than would be expected if the effects of α2AP-bindingmolecule administration and thrombolytic agent administration was merelyadditive.

[0112] The α2AP-binding molecule of the present invention encompassesclot-specific inhibitors of α2AP. It is demonstrated that thecombination of t-PA and the specific inhibitors, particularly monoclonalantibodies to α2AP, does not increase fibrinogen consumption over thatobtained with equipotent doses of plasminogen activator alone. Further,clot-specific inhibition of α2AP enhances the potency of plasminogenactivators and preserves fibrin selectivity. For agents such asurokinase, which is not selective for fibrin, inhibition of clot boundα2AP would cause a similar, or more pronounced, enhancement in potencyand lead to less fibrinogen consumption as well.

[0113] Thus, the inhibition of clot-bound α2AP enhances clot lysis byendogenous plasminogen activators. Further, when administered incombination with a thrombolytic agent, thrombolysis is significantlyincreased compared with the lysis achieved by equivalent doses of thethrombolytic agent alone. This increased lysis S by the combination ofthe thrombolytic agent and α2AP inhibitor occurs without decreasingcirculating fibrinogen or α2AP levels. The net result is a synergisticinteraction between the two agents.

[0114] When used alone, an amount of α2AP-binding molecule capable ofpreventing inhibition of plasmin and thereby enhancing clot lysis whenprovided to a patient is a “therapeutically effective” amount. In orderto enhance clot lysis and prevent clot reformation, it is desirable toprovide between 3 to 300 nmole of α2AP-binding molecule per kilogram ofpatient weight. This dosage may be administered, in one embodiment, overa period of between 60 to 480 minutes, by continual intravenous infusionat a rate of 0.006 to 5 nmole/kg/min. Alternatively, it is possible toprovide the α2AP-binding molecule in an intravenously injectable bolusat a dose of between 3 to 600 nmole/kg, and most preferably between 30to 60 nmole (of α2AP-binding molecule) per kilogram of patient weight.If the α2AP-binding molecule is provided in this manner, a single bolusis sufficient to prevent potential clot reformation. The α2AP-bindingmolecule of the present invention may be dissolved in anyphysiologically tolerated liquid in order to prepare an injectablebolus. It is preferable to prepare such a bolus by dissolving theα2AP-binding molecule in normal saline.

[0115] When the α2AP-binding molecule capable of preventing inhibitionof plasmin is co-administered with a thrombolytic agent, it is desirableto provide 3 to 300 nmole of α2AP-binding molecule per kilogram ofpatient weight. This dosage may be administered, in one embodiment, overa period of 60 to 480 minutes, by continuous intravenous infusion.Alternatively, it is possible to provide the α2AP-binding molecule in anintravenously injectable bolus at a dose of between 3 to 600 nmole/kg,and most preferably between 30 to 60 nmole/kg of patient weight. Anamount of thrombolytic agent capable of causing such lysis is a“therapeutically effective” amount. It is desirable to provide between0.01 to 3.0 mg per kilogram of patient weight. In one embodiment, thethrombolytic agent is provided over a prolonged period (i.e., from about180 to about 1440 minutes). In a preferred embodiment, the thrombolyticagent of the present invention is provided as an intravenously injectedbolus containing between 0.5 to 1.0 mg/kg, and most preferably between0.5 to 0.75 mg/kg. For example, for pulmonary embolism, the dosage oft-PA by continuous infusion is ˜100 mg for 2 hours (Goldhaber, S. C. etal., Lancet 341:507 (1993)). The dosage to be used of thrombolytic agentof the present invention is generally known in the art (see, e.g.,Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rded. Philadelphia, Pa. (1994)).

[0116] The thrombolytic agent of the present invention may be dissolvedin any physiologically tolerated liquid in order to prepare aninjectable bolus. It is, however, preferable to prepare such a bolus bydissolving the thrombolytic agent in normal saline.

[0117] A patient treated according to the preferred embodiment will,therefore, receive an intravenously injected bolus of the α2AP-bindingmolecule in combination with an intravenously injected bolus of thethrombolytic agent. This preferred treatment minimizes the amount oft-PA required for thrombolysis, thus reducing the extent of fibrinogenbreakdown and lessening any tendency for general hemorrhage.Importantly, the use of the preferred treatment results in thedissolution of the occluding thrombus at a rate which greatly exceedsthe rate of thrombus dissolution when either the α2AP-binding moleculeor the thrombolytic agent is provided by infusion alone. Additionally,the risk of reocclusion is substantially reduced.

[0118] In previous models of fibrinolysis (3), the chief role assignedto α2AP has been to inactivate circulating plasmin and prevent asystemic lytic state. Thus, it may be surprising that an α2AP inhibitorcan increase clot lysis without increasing fibrinogenolysis. This markedamplification of thrombolysis by α2AP inhibitor underscores theimportance of fibrin bound α2AP in regulating fibrinolysis. Since thesubject antibodies augment clot lysis by a fibrin-selective agent suchas t-PA as well as that by the nonselective activators urokinase andstreptokinase, it appears that fibrin-bound α2AP plays a critical rolein determining the rate of lysis by any exogenous plasminogen activator.

[0119] These unexpected findings are important because it had previouslynot been possible to accelerate the rate of clot lysis withoutincreasing the tendency to hemorrhage. The preferred embodiment,therefore, provides a method of treatment in which the administration ofa bolus of a α2AP-binding molecule in combination with theadministration of a bolus of a thrombolytic agent are capable ofdissolving an occluding thrombus at a faster rate than can be obtainedwhen either compound is administered alone. Moreover, the preferredembodiment accomplishes this goal while minimizing both fibrinogenbreakdown and the risk of reocclusion. Thus, the combination of agentscan significantly increase the potency and specificity of thrombolytictherapy.

[0120] As would be apparent to one of ordinary skill in the art, therequired dosage of the anti-α2AP binding molecule or thrombolytic agentwill depend upon the severity of the condition of the patient, and uponsuch criteria as the patient's height, weight, sex, age, and medicalhistory.

[0121] The α2AP-binding molecule or thrombolytic agent of the presentinvention can be formulated according to known methods to preparepharmaceutically useful compositions, such as by admixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation are described, for example, in Remington's PharmaceuticalSciences, 16th Ed., Osol, A., ed., Mack, Easton Pa. (1980). In order toform a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe α2AP-binding molecule or thrombolytic agent, either alone, or with asuitable amount of carrier vehicle.

[0122] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the α2AP-binding molecule orthrombolytic agents of the present invention. The controlled deliverymay be exercised by selecting appropriate macromolecules (for example,polyesters, polyamino acids, polyvinyl pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate). The rate of drug release may also be controlled byaltering the concentration of such macromolecules. Another possiblemethod for controlling the duration of action comprises incorporatingthe therapeutic agents into particles of a polymeric substance such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. Alternatively, it is possible to entrap thetherapeutic agents in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences, 16thEd., Osol, A., ed., Mack, Easton Pa. (1980).

[0123] The thrombolytic agent or α2AP-binding molecule may be providedto a patient by means well known in the art. Such means of introductioninclude oral means, intranasal means, subcutaneous means, intramuscularmeans, intravenous means, intra-arterial means, or parenteral means. Inone preferred method of treatment for pulmonary embolism, myocardialinfarction, thrombosis or stroke, a patient is provided with a bolus(intravenously injected) containing between 0.5 to 1.0 mg/kg of athrombolytic agent.

[0124] Generally, the results reported herein demonstrate that aninhibitor, particularly a monoclonal antibody, can be used to augmentthe catalytic function of an enzyme by neutralizing an inhibitor of thatenzyme. This approach can be applied to biological processes which aretightly governed by inhibitors. Because coagulation is a finely balancedsystem in which the effects of enzymes (generally serine proteases) arepitted against the effects of inhibitors, frequently serpins (serineprotease inhibitors) pathological alterations in clotting can be treatedby augmenting enzyme activity or by neutralizing an inhibitor.

[0125] Having now generally described this invention, the same will bebetter understood by reference to certain specific examples which areincluded herein for purposes of illustration and are not intended aslimiting.

EXAMPLE 1

[0126] Preparation of an Antibody Directed to Alpha-2-antiplasmin

[0127] A. Monoclonal Antibody Production, Purification andCharacterization

[0128] Two Balb/C mice were immunized subcutaneously with 25 μg ofpurified human α2AP fragments derived from the trypsin digest of a humanplasma clot. The α2AP fragments were affinity purified with aSEPHAROSE-coupled monoclonal antibody, RWR (Reed, G. L. III et al.,Trans. Assoc. Am. Phys. 101:250-256 (1988); U.S. Pat. No. 5,372,812,issued Dec. 13, 1994), against human α2AP. Mice were initially immunizedwith complete Freund's adjuvant and boosted 90 days later with 50 μg ofα2AP fragment in incomplete Freund's adjuvant. The antisera titer wastested in a solid-phase radioimmunoassay (Reed, G. L. III et al., Proc.Natl. Acad. Sci. USA 87:1114-1118 (1990)) with α2AP immobilized in thewells of a microtiter plate. Four days before fusion, the mouse with thehighest titer of α2AP antibody was hyperimmunized with 100 μg α2APintraperitoneally. Somatic cell fusion was performed as described(Galfre, G. and Milstein, C., Meth. Enzymol. 73:3-46 (1981)).

[0129] Hybridomas were tested for the production of antibodies to theα2AP fragment and for their ability to inhibit α2AP as described inReed, G. L. III et al., Proc. Natl. Acad Sci. USA 87:1114-1118 (1990).The binding of monoclonal antibodies (MAbs) to ¹²⁵I-α2AP was tested in asolid-phase radioimmunoassay. Wells of a microtiter plate were coatedwith goat antimouse antibody (25 μl, 5 μg/ml) for 2 hours. The wellswere rinsed and nonspecific protein binding sites were blocked with 1%bovine serum albumin in Tris-buffered saline, pH 7.4, for 1 hour. Aftera wash, 25 μl of hybridoma supernatant was added to the wells andincubated for 1 hour. The wells were rinsed and ¹²⁵I-α2AP was added (25μl, 60,000 cpm) for 1 hour. The ¹²⁵I-α2AP was then removed and the wellswere rinsed and gamma-counted.

[0130] Cloned hybridomas (limiting dilution) were expanded into ascitesin pristane-primed Balb/C mice. Antibodies were purified from filteredascites by precipitation with 40% ammonium sulfate, dialysis into 10 mMKH₂PO₄, pH 7.2, and ion-exchange chromatography on DEAE-AFFIGEL BLUESEPHAROSE (BioRad, Hercules, Calif.) with a linear gradient from 0 to100 mM NaCl.

[0131] B. Immunoblotting

[0132] These were performed largely as described in Reed, G. L. et al.,J. Immunol. 150:4407-4415 (1993). Purified human α2AP (5 μg, AmericanDiagnostica, Greenwich, Conn.) was electrophoresed in a large singlesample lane on a 12% slab minigel (BioRad, Hercules, Calif.) underreducing and non-reducing conditions. The sample was electroblotted(Kyhse-Anderson, 1084) to polyvinylidene difluoride membranes(Millipore, Bedford, Mass.) and nonspecific protein binding sites wereblocked with 5% dry milk. The blots were exposed to different hybridomassupernatants for 1 hour in the channels of a miniblotter apparatus(Immunetics, Cambridge, Mass.). After washing, the bound antibody wasdetected by incubation with ¹²⁵I-goat antimouse antibody (1.5 millioncpm/membrane). After additional washing, the membranes were exposed in aphosphorimager (Molecular Devices, Sunnyvale, Calif.).

[0133] C. Radioimmunoassays

[0134] Wells of a microtiter plate were coated with goat antimouseantibody (25 μl, 5 μl/ml) for 2 hours at 21° C. They were washed andblocked with 1% BSA (bovine albumin serum) for 1 hour. To the emptywells in duplicate were added 25 μl of hybridoma supernatants. Afterincubation for 1 hour the wells were washed and 25 μl of ¹²⁵I-α2AP wasadded to the wells for another hour. After washing the wells were cutand the bound antibody measured by gamma scintillation counting.

[0135] Competition radioimmunoassays were performed by coating wells ofa microtiter plate with 25 μl of purified MAb (70B11) in duplicate (10μg/ml) for 1 hour. The wells were washed and blocked with 1% BSA for 1hour. After washing, 25 μl of a competitor MAb, same MAb or negativecontrol MAb was added to different wells (50 μg/ml) followed by 25 μl of¹²⁵I-α2-antiplasmin (100,000 cpm). After 1 hour incubation, the wellswere washed, cut and the radioactivity was measured in a gammascintillation counter.

[0136] D. Plasma Clot Lysis Assays

[0137] These were performed largely as described in Reed, G. L. III etal., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990). Pooled fresh frozenplasma was obtained from 5 random donors to the Massachusetts GeneralHospital Blood Bank. The plasma was mixed with ¹²⁵I-fibrinogen toachieve ˜1,000 cpm/μl. The plasma was clotted for 1 hour at 37° C. in a12×65 mm test tube by mixing 50 μl with 50 μl of CaCl₂ (5 mM final) andthrombin (1 U/ml). Clots were compressed and washed in 1 mlTris-buffered saline (pH 7.4) to remove unbound proteins. Thesupernatant was removed and the clots were counted in a gamma counter.To each set of duplicate clots was added 100 μl containing variousamounts of urokinase, 100 μl of pooled plasma containing 1 u/ml ofhirudin and 100 μl of Tris-buffered saline containing 7 μg (FIG. 4) or21 μg (FIG. 5) of MAb or no MAb. The clots were placed in a 37° C. waterbath and at various intervals 100 μl of solution was temporarily removedand gamma-counted to determine the amount of lysis. The percent lysiswas defined at 100× (total supernatant cpm÷total clot cpm).

[0138] E. Results

[0139] Three hybridomas were selected that appeared to inhibit α2APfunction in screening assays. The serotypes of these MAbs were: 49C9(Igγ2aK), 70B11 (Igγ1K), and 77A3 (Igγ2aK). FIG. 1 compares the bindingof these MAbs to ¹²⁵I-α2AP in a reverse solid-phase assay. When comparedto the original α2AP inhibitor RWR, these MAbs bound with greateravidity. To determine if the MAbs bound to the same epitopes,competition assays is shown for 70B11 in FIG. 2. Compared to thenegative control, anti-digoxin MAb, RWR had no significant inhibitoryeffects on the binding of ¹²⁵I-α2AP to immobilized 70B11. In contrast,when 70B11 was used as a competitor, it completely inhibited the bindingof ¹²⁵I-α2AP to immobilized 70B11, as expected. However, 49C9 and 77A3were also excellent competitors as well. The results of these assays areshown in tabular form in Table 1, below. MAbs 49C9, 70B11, 77A3 alsofully inhibited the binding of each other to ¹²⁵I-α2AP, but they had noinhibitory effects on the binding of RWR. The converse was also true,RWR as a competitor had no effect on the binding of ¹²⁵I-2AP to theother MAbs. This indicated that MAbs 49C9, 70B11 and 77A3 competed forbinding to the same epitope, while RWR appeared to bind to a separateregion of α2AP.

[0140] To determine if the MAbs recognized a continuous or discontinuousepitope in α2AP, immunoblotting experiments were performed withdenatured and reduced α2AP. In these studies RWR bound well to denaturedand reduced α2AP, indicating that it recognized an epitope which was notaffected by boiling with SDS, nor reduction of disulfide bonds. Incontrast, MAbs 49C9, 70B11 and 77A3 did not bind to denatured α2AP,suggesting that they recognize a conformation-dependent epitope.

[0141] Clot lysis assays were performed to examine the relative potencyof these MAbs in amplifying the fibrinolysis by urokinase. FIG. 3compares the amount of lysis achieved by 7 μg of different purified MAbs(or TBS alone) as a function of dose of urokinase. Compared to urokinasealone (TBS) or urokinase with the control antidigoxin MAb, RWR, 49C9,70B11 and 77A3 all accelerate clot lysis. However, 49C9, 70B11 and 77A3appear to be significantly more potent than RWR in these assays. Toexamine the increase in fibrinolytic potency of urokinase achieved byone of these antibodies, dose response studies were performed in theabsence or presence of MAb 77A3. FIG. 4 shows that MAb 77A3 markedlyincreases the potency of lysis of urokinase by roughly 100-fold.

[0142] As a means of further discriminating among the functional andepitope binding specificities of these MAbs, their ability to inhibitthe α2AP from different animal species in plasma clot lysis assays wasexamined. The results of these assays are summarized in Table 2, below.In the different species of animal plasmas tested, RWR appeared toinhibit only human α2AP. In contrast, the other MAbs showed a broaderspecies cross-reactivity and ability to inhibit nearly all primate andsome non-primate α2APs. TABLE 1 Effect of Different MAb Inhibitors onthe binding of ¹²⁵I-α2AP to Immobilized MAbs. Immobilized MAb InhibitorRWR 49C9 70B11 77A3 RWR + − − − 49C9 − + + + 70B11 − + + + 77A3 − + + +anti-digoxin − − − −

[0143] TABLE 2 The cross reactivity of MAbs with differentα2-antiplasmins. Species RWR 49C9 70B11 77A3 HUMAN ++ ++ ++ ++ Baboon −++/+ ++/+ ++ Grn Monkey − ++ ++ ++ Dog − + +/− + cynamologus − ++ ++ ++marmoset − + + + ferret − +/++ +/− +/++ guinea pig − − +/− +/− gerbil −− − −

EXAMPLE 2 In Vivo Study of Pulmonary Embolism

[0144] A. Materials

[0145] Materials were obtained from the following suppliers: rt-PA witha specific activity of 580,000 IU/mg, Genentech (South San Francisco,Calif.); ketamine (100 mg/ml), Fort Dodge Laboratories (Fort Dodge,Iowa); acepromazine maleate, Fermenta Animal Health Co. (Kansas City,Mo.); heparin (1000 U/ml), Elkins-Sinn Inc. (Cherry Hill, N.J.); sodiumiodide, Aldrich Chemical Co. (Milwaukee, Wis.); calcium chloride,Mallinckrodt (Paris, Ky.); normal saline for intravenous use, TravenolLaboratories (Deerfield, Ill.); α2AP assay kit, Stachrom (Asnières,France); purified α2AP and fibrinogen, American Diagnostica (Greenwich,Conn.); goat antimouse antibody, Cappel Organon Technika (Durham, N.C.);human plasma pooled from random donors, Massachusetts General Hospital(Boston); bovine thrombin, Parke-Davis (Morris Plains, N.J.); Na¹²⁵I,Dupont-NEN (Cambridge, Mass.); Bard Parker surgical blade, BectonDickinson (Franklin Lake, N.J.); 4.0 silk sutures, American Cyanamid Co.(Danbury, Conn.); SURFLO IV catheter and 20 gauge 1¼-inch VENOJECT tubeswith K₃EDTA, Terumo Medical Corp. (Elkton, Md.); sterile three-waystopcock, Mallinckrodt Critical Care (Glens Falls, N.Y.); auto syringeinfusion pump, Baxter Health Care Corp. (Hooksett, N.H.); infusion pumptubing and microbore 60-inch extension set, McGaw of Puerto Rico (SabanaGrand, Puerto Rico); surgical instruments, VWR (Boston); tubing, Namic(Glens Falls, N.Y.); ferrets (˜0.8-1 kg), Marshall Farms (New York,N.Y.); aprotinin, Sigma (St. Louis, Mo.); and microcentrifuge tubes,National Scientific Supply Co. (San Rafael, Calif.).

[0146] B. In Vitro Clot Lysis Assays

[0147] Pooled, fresh-frozen, citrated ferret plasma (1100 μl) was mixedwith 15 μl of ¹²⁵I-labeled human fibrinogen (˜40,000 cpm/clot). Ferretplasma (35 μl) was mixed with 35 μl of Tris-buffered saline (TBS)containing 10 nM CaCl₂ and thrombin (1 U/ml) in twelve 65-mm plastictubes and clotted for 1 hour at 37° C. The clots were washed in TBS, thesupernatant was removed, and then 100 μl of TBS or 25 μg of purified MAb(RWR or 77A3) was added to tubes in duplicate. Clot lysis was initiatedby adding 0.1 U of rt-PA per tube. The clots were incubated at 37° C.for 5 hours and the amount of lysis was determined by sampling for therelease of radiolabeled fibrin degradation products into thesupernatant, as described (Reed, G. L. III et al., Proc. Natl. Acad.Sci. USA 87:1114-1118 (1990)).

[0148] C. Pulmonary Embolism Experiments

[0149] Male ferrets were anesthetized by intramuscular injection (0.4ml) of a mixture of ketamine and acepromazine (two parts acepromazine[10 mg/ml] to one part ketamine [100 mg/ml]). Intraperitoneal injectionswere repeated as necessary to keep the animals anesthetized. After ananterior midline incision had been made in the neck, the jugular veinand the carotid artery were exposed by blunt dissection and cannulatedwith 20G catheters that were secured at the proximal and distal endswith 4-0 silk sutures. The catheters were capped with three-waystopcocks.

[0150] Pooled, citrated human plasma was mixed with ¹²⁵I-fibrinogen toachieve ˜1,000,000 cpm/ml. Individual clots were formed by mixing¹²⁵I-fibrinogen-labeled plasma (45 μl) with 2.5 μl of bovine thrombin(100 U/ml) and 2.5 μl of calcium chloride (0.4 M). These clots wereincubated at 37° C. for 90 minutes, compressed, and washed thoroughlywith saline three times to remove unbound proteins. The radioactivecontent of the clots was measured in a gamma counter immediately beforeclot injection. Blood samples were drawn at base line and at the end ofthe experiment. Sodium iodide (10 mg) was injected to block thyroiduptake. Clots were embolized into the lungs by injection through theinternal jugular vein. Ferrets weighing less than 1 kg received threeclots; those weighing 1 kg or more received four clots. Successfulembolization was evidenced by the accumulation of radioactivity in thethorax. After the clots had been injected, the ferrets were turned ontheir sides to ease breathing.

[0151] All animals received weight-adjusted heparin at 100 U/kg (bolus),a dose sufficient to keep the activated partial thromboplastin time(aPTT) above 150 seconds throughout the procedure. The α2AP inhibitor(sterile-filtered, 14 mg/ml in saline) or a purified control MAb(antidigoxin) was given intravenously as a single dose (22.5 mg/kg). Thert-PA was given as a continuous infusion over 2 hours (1 or 2 mg/kg in 5ml normal saline). Animals were observed for a total of four hours afterpulmonary embolization and then killed by lethal injection of anesthesiaor by CO₂ inhalation. The thorax was dissected and all intrathoracicstructures were removed for gamma counting to detect residual thrombi.The percentage of clot lysis was determined for each ferret by dividingthe total residual radioactivity in the thorax (cpm) by that in theinitial thrombi.

[0152] This experimental protocol was approved by the Harvard MedicalArea Standing Committee on Animals. The Harvard Medical School animalmanagement program is accredited by the American Association ofLaboratory Animal Care, and the procedures were conducted in accordancewith National Institutes of Health standards, as set forth in the Guidefor the Care and Use of Laboratory Animals (DHHS Publication No. [NIH]85-23, revised 1985), the Public Health Service Policy on the HumaneCare and Use of Laboratory Animals by Awardee Institutions, and the NIHPrinciples for the Utilization and Care of Vertebrate Animals Used inTesting, Research, and Training.

[0153] D. Statistical Tests

[0154] The data were analyzed by a one way analysis of variance followedby a Bonferroni-Dunn procedure for multiple comparison testing.

[0155] E. Fibrinogen Assays

[0156] Blood samples were collected on K₃EDTA (0.15% solution final)with aprotinin (50 kallikrein U/ml). Platelet-poor plasma was obtainedby centrifugation of whole blood (Mustard, J. F. et al., Meth. Enzymol.169:3-11 (1989)) and assayed for fibrinogen by the sodium sulfite method(Rampling, M. W. and Gaffney, P. J., Clin. Chim. Acta. 67:43-52 (1976)).

[0157] F. α2-Antiplasmin Assays

[0158] To measure α2AP levels, we collected ferret blood on sodiumcitrate (1/10 volume) and centrifuged it to obtain plasma (Mustard, J.F. et al., Meth. Enzymol. 169:3-11 (1989)). The plasma was tested forfunctional α2AP with a chromogenic substrate assay for plasmininhibition (Stachrom kit) as described (Reed, G. L. III et al., Proc.Natl. Acad. Sci. USA 87:1114-1118 (1990)).

[0159] G. Results

[0160] From a panel of hybridomas we selected 77A3, a MAb that boundtightly to human α2AP. MAb 77A3 was purified from mouse ascites by ionexchange chromatography, and its purity was confirmed bySDS-polyacrylamide gel analysis (FIG. 5). To study the role of α2AP inexperimental pulmonary embolism in vivo, we tested purified 77A3 inseveral different animal plasma clot lysis assays to determine whetherit could bind and inhibit a non-human α2AP. Of various small animalplasmas tested (e.g. hamster, gerbil, guinea pig, rat, etc.), 77A3significantly crossreacted with ferret plasma. FIG. 6 compares the lyticeffects of 77A3 with those of another MAb inhibitor of human α2AP, RWR(Reed, G. L. III et al., Trans. Assoc. Am. Phys. 101:250-256 (1988);U.S. Pat. No. 5,372,812, issued Dec. 13, 1994), and with buffer alone.FIG. 6 shows that in comparison with the control (buffer alone), 77A3accelerated the lysis of ferret plasma clots induced by a low dose ofrt-PA (0.1 unit). In contrast, RWR, which inhibits human α2AP (Reed, G.L. III et al., Trans. Assoc. Am. Phys. 101:250-256 (1988); U.S. Pat. No.5,372,812, issued Dec. 13, 1994) but does not crossreact with nonhumanα2AP, had no detectable effect. This experiment indicated that 77A3inhibited ferret α2AP and amplified ferret clot lysis in vitro.

[0161] The cross-reactivity of 77A3 allowed us to investigate the roleof α2AP in a ferret model of pulmonary embolism. In humans, pulmonaryembolism is usually treated with heparin (Goldhaber, S., Chest107:45S-51S (1995)). Consequently, ferrets were treated with aweight-adjusted bolus dose of heparin at 100 U/kg. This dose wassufficient to keep the aPTT above 150 seconds throughout the experiment(n=3). To investigate the effects of intravenous MAb 77A3 on theactivity of α2AP in the blood, we selected a dose, 22.5 mg/kg, that wasin molar excess to the level of ferret α2AP. Our ex vivo measurements offerret α2AP activity, 1 and 4 hours after intravenous dosing, showedthat ˜75% of ferret α2AP activity was inhibited at this dose (FIG. 7,n=2).

[0162] Using heparin at 100 U/kg and 77A3 at 22.5 mg/kg, we theninvestigated the effect of these agents and rt-PA on the lysis ofpulmonary emboli (FIG. 8). All animals received heparin. Control animals(n=8), which received no rt-PA, showed 15.6±10.5% (mean±SD) lysis oftheir pulmonary emboli. Animals receiving rt-PA at 1 mg/kg (n=4) over 2hours showed 38.5±6.3% lysis, which was significantly greater than lysisobtained in those receiving heparin alone (P<0.01). Similarly, animalsreceiving rt-PA at 1 mg/kg and a control (antidigoxin) MAb (n=3) showed35.2±4.6% lysis. Ferrets treated with rt-PA at 2 mg/kg (n=4) showed aminimal increase in lysis over those treated at 1 mg/kg (45.0±6.5% vs38.5±6.3%, P<0.05). However, animals receiving rt-PA at 1 mg/kg togetherwith the α2AP inhibitor (n=4) showed greater lysis (56.2±4.7%) thanthose receiving an equivalent dose of rt-PA alone (P<0.01), with orwithout the control (antidigoxin) MAb (P<0.01), or those receiving twicethe dose of rt-PA alone (P<0.05).

[0163] In addition to inhibiting plasmin on the thrombus surface, α2APand other inhibitors inactivate plasmin in the blood (Collen, D., Eur.J. Biochem. 69:209-216 (1976); Moroi, M. and Aoki, N., J. Biol. Chem.251:5956-5965 (1976); Mullertz, S. and Clemmensen, I., Biochem J.159:545-553 (1976)). We measured fibrinogen levels in the blood todetermine if inhibition of α2AP led to nonspecific plasminolysis of acirculating clotting factor. FIG. 9 shows residual fibrinogen levelsexpressed as a function of their initial values in four treatmentgroups. In animals that received no rt-PA, fibrinogen levels variedmoderately but did not diminish during the experiment. Ferrets receiving1 mg/kg and 2 mg/kg of rt-PA alone showed no significant change infibrinogen level. Similarly, animals receiving the combination of rt-PAand the α2AP inhibitor showed no detectable change in circulatingfibrinogen levels.

[0164] H. Discussion

[0165] Clinical and experimental studies suggest that pulmonary emboliand venous thrombi resist endogenous fibrinolysis and lysis induced byplasminogen activators (Goldhaber, S., Chest 107:45S-51S (1995);Goldhaber, S. Z. et al., Lancet 2:886-889 (1986); The UrokinasePulmonary Embolism Trial, Circulation 47:1-108 (1973); Goldhaber, S. Z.et al., Am. J. Med. 88:235-240 (1990); Goldhaber, S. Z. et al., Lancet341:507-511 (1993)). This resistance to lysis is due in part to specificmolecular factors in the thrombus that act to oppose fibrinolysis.During thrombus formation, α2AP is covalently crosslinked to fibrin byactivated factor XIII (Sakata, Y. and Aoki, N., J. Clin. Invest.69:536-542 (1982)). Studies in vitro indicate that when α2AP in the clotis absent or inhibited by MAbs, clots undergo spontaneous lysis (Aoki,N. et al., Blood 62:1118-1122 (1983); Miles, L. A et al., Blood59:1246-1251 (1982); Reed, G. L. III et al., Trans. Assoc. Am. Phys.101:250-256 (1988); Reed, G. L. III et al., Proc. Natl. Acad. Sci. USA87:1114-1118 (1990)). Conversely, when levels of α2AP in clots areincreased by supplementation in vitro, fibrinolysis is inhibited(Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982)). In thepresent study we investigated the hypothesis that α2AP plays a majorregulatory role in fibrinolysis and that it contributes to the thrombusresistance obtained in pulmonary embolism.

[0166] We measured the effect of rt-PA, with and without α2APinhibition, on the net lysis of pulmonary emboli in ferrets. Becauseheparin is the established therapy for humans with pulmonary embolism,we considered animals treated with heparin alone as the control group.The weight-adjusted bolus dose of heparin given to the ferrets wassufficient to maintain a high level of anticoagulation throughout theexperiment. In animals treated with rt-PA, at a dose comparable to thatused in humans (1 mg/kg), lysis of pulmonary emboli was enhancedsignificantly in comparison with lysis in animals treated with heparinalone. Increasing the dose of rt-PA to 2 mg/kg, a dose higher than issafe in humans, led to a minimal increase in lysis. A similar plateau inthe dose response for t-PA-induced lysis has been noted in experimentalstudies of pulmonary embolism in dogs (Werier, J. et al., Chest.100:464-469 (1991)). However, specific inhibition of α2AP markedlypotentiated the lysis of experimental pulmonary emboli by rt-PA (1mg/kg), causing significantly more lysis than was seen in ferretstreated with the same dose of rt-PA: alone or with a control MAb, thelysis achieved with α2AP inhibition was also greater than that achievedin ferrets treated with high-dose rt-PA (2 mg/kg). At the same time,despite the higher total lysis obtained in animals treated with the α2APinhibitor, there was no significant consumption of circulatingfibrinogen. In these studies of experimental pulmonary embolism, α2APplayed an important role in thrombus resistance to lysis induced byrt-PA. Further studies will be necessary to establish the relativequantitative roles of circulating and thrombus bound α2AP in thisprocess.

[0167] Besides α2AP, other molecular factors may regulate the thrombusresistance of pulmonary emboli. A leading candidate is PAI-1, a serineprotease inhibitor of t-PA and urinary-type plasminogen activator (u-PAor urokinase) (Stringer, H. A. and Pannekoek, H., J. Biol. Chem.270:11205-11208 (1995); Carmeliet, P. et al., J. Clin. Invest.92:2756-2760 (1993); Lang, I. M. et al., Circulation 89:2715-2721(1994); Marsh, J. J. et al., Circulation 90:3091-3097 (1994)). Unlikeα2AP, PAI-I is not specifically crosslinked to fibrin in the thrombus,although it has been shown to bind to fibrin in vitro (Stringer, H. A.and Pannekoek, H., J. Biol. Chem. 270:11205-11208 (1995)). By addingrecombinant PAI-1 to developing thrombi, Marsh et al. (Marsh, J. J. etal., Circulation 90:3091-3097 (1994)) have shown that PAI-1-enrichedclots can suppress the spontaneous lysis of pulmonary emboli in a caninemodel; however, the role of PAI-1 in the lysis of autologous thrombi wasnot investigated. Pathologic studies of pulmonary emboli extracted bythrombectomy have suggested that PAI-1 expression increases in theendothelial cells at the margins of fresh thrombi but is not detectablein the thrombi themselves (Lang, I. M. et al., Circulation 89:2715-2721(1994)). Since PAI-1-deficient mice (by gene deletion) are less likelythan regular mice to develop venous thrombosis induced by endotoxin(Carmeliet, P. et al., J. Clin. Invest. 92:2756-2760 (1993)), theexpression of PAI-1 in endothelial cells at the margin of the developingthrombus may be functionally important. Nonetheless, the role of PAI-1in thrombus resistance to pharmacologic plasminogen activators is lessclear: in patients given t-PA, the inhibitory capacity of PAI-1 isoverwhelmed completely (Lucore, C. L. and Sobel, B. E., Circulation77:660-669 (1988)), and thrombus resistance is also observed in patientsgiven streptokinase, against which PAI-1 has no effect.

[0168] Another potential cause of thrombus resistance in pulmonaryembolism is activated factor XIII. Several studies in vitro suggest thatthis coagulation enzyme renders the fibrin in clots more resistant todegradation by plasmin by crosslinking fibrin chains together and bycrosslinking α2AP to fibrin. (Sakata, Y. and Aoki, N., J. Clin. Invest.69:536-542 (1982); Robbie, L. A. et al., Thromb. Haemostas. 70:301-306(1993); Francis, C. W. and Marder, V. J., J. Clin. Invest. 80:1459-1465(1987); Jansen, J. W. C. M. et al., Thromb. Haemostas. 57:171-175(1987); Reed, G. L. et al., Trans. Assoc. Am. Phys. 104:21-28 (1991))However, little is known about activated factor XIII and thrombusresistance in vivo. This is probably due to the fact that a potentinhibitor of factor XIII function has only recently become available(Reed, G. L. and Lukacova, D., Thromb. Haemostas. 74:680-685 (1995)).One study has suggested that when factor XIII is partially inhibited,coronary thrombi lyse at accelerated rates in response to t-PA(Shebuski, R. J. et al., Blood 75:1455-1459 (1990)). This observationargues that factor XIII, through its effects on fibrin-fibrin andα2AP-fibrin crosslinking, also contributes to thrombus resistance.

[0169] Improving the lysis of thrombi in patients with pulmonaryembolism and deep venous thrombosis remains a challenge. Unfortunately,increasing the dose of plasminogen activators is not a promisingapproach. High dose t-PA has been associated with an unacceptableincrease in the risk of cerebral bleeding (Passamani, E. et al., J. Am.Coll. Cardiol. 10:51B-64B (1987)). In addition, in the present study andothers (Werier, J. et al., Chest. 100:464-469 (1991)), high-dose t-PA(≧2 mg/kg) produced only minimal increases in net lysis. The currentFDA-approved doses of urokinase and streptokinase cause plasminogen“depletion”; thus, increasing the doses of these agents is also notlikely to have an effect on net lysis (Onundarson, P. T. et al., J. Lab.Clin. Med. 120:120-128 (1992)). Several potent inhibitors of thrombingeneration and activity are under development. Although these agents mayfurther reduce the formation of new thrombi, they will not directlyimprove lysis of the large thrombi that typically exist in patients atthe time they are diagnosed. These considerations suggest thatfundamental insights into the molecular factors that oppose physiologicor pharmacologic lysis in thrombi will be necessary to spark improvedtreatments for venous thromboembolism. The results of the present studyindicate that α2AP is a major contributor to thrombus resistance inexperimental pulmonary embolism, and they suggest that inhibiting α2APmight improve lysis in patients with thrombotic disease.

EXAMPLE 3 Cloning and Sequencing of Antibody cDNA

[0170] A. Amino Terminal Sequences of Antibodies

[0171] Monoclonal antibodies (49C9, 70B11 and 77A3) were expanded intoascites and purified by ion exchange chromatography on DEAE Affigel Blueor by protein A agarose as described in Lukacova, D. et al.,Biochemistry 30:10164-10170 (1991). The purified MAbs (15 μg) weresubjected to SDS-PAGE on 10% minigels (BioRad, Hercules, Calif.). Theprotein samples were electroblotted to PVDF membranes (Millipore,Bedford, Mass.) using semi-dry technique (Kyhse-Anderson, J., J.Biochem. Biophys. Meth. 10:203-209 (1984)) at 4° C. for 2 hrs at 75milliamps (Millipore electroblotter). The bands were stained withPonceau Dye (Sigma, St. Louis) and excised. The amino terminal sequencesof the light chain of the antibodies are shown in FIG. 10 (SEQ ID NOS:1-3).

[0172] B. Molecular Cloning of Antibody cDNA

[0173] Cloned hybridoma cell lines 49C9, 70B11 and 77A3 were grown in150 mm tissue culture plates in 20% fetal bovine serum in Dulbecco'smodified Eagle's medium with 4.5 g/l of glucose and penicillin andstreptomycin. The cells were harvested and centrifuged at 1200 rpm for 7min. The cell pellet was resuspended in sterile phosphate bufferedsaline (pH 7.4) and re-centrifuged. Then 5 ml of RNAzol (Teltest,Friendswood, Tex.) was added and the pellet was homogenized for 2 min.Chloroform (500 μl) was added and the mixture was vortexed and left toincubate on ice for 15 min. The samples were centrifuged at 12,000 rpmfor 15 min. The aqueous layer was mixed with 4.5 ml of isopropanol andvortexed. The mixture was precipitated at −70° C. for 90 min. andrecentrifuged at 12,000 rpm for 15 min. The pellet was washed in 2 ml of70% ethanol in DEPC-treated water. After repeat centrifugation, thesupernatant was removed and the pellet air-dried. The pellet wasdissolved in 200 μl of diethyl-pyrocarbonate (DEPC)-treated water and 20μl of 3 M NaCl and 800 μl of ethanol were added. The mRNA wasprecipitated overnight at −70° C. and the pellet resuspended inDEPC-water.

[0174] The cDNA corresponding to the light and heavy chain sequenceswere isolated by primer guided reverse transcription followed bypolymerase chain reaction as described (Gene Amp Thermostable rTthReverse Transcriptase RNA PCR kit (Perkin-Elmer Cetus, San Francisco,Calif.). The light chain mRNA was primed for reverse transcription witha 3′ primer (5′ N6GAATTCACTGGATGG TGGGAAGATGGA 3′ (SEQ ID NO: 22))corresponding to the constant region of the light chain (Coloma, M. J.,et al., Biotechniques 11:152-154, 156 (1991)) and the heavy chain wasprimed with a 3′ primer (5′ N6GAATTCA(TC)CTCCACACACAGG(AG)(AG)CCAGTGGATAGAC 3′ (SEQ ID NO: 23)) corresponding tothe constant region of the heavy chain (Coloma, M. J., et al.,Biotechniques 11:152-154, 156 (1991)). Because the light chain aminoterminal sequences were known, a specific primer corresponding to thelikely 5′ sense sequence was used (5′ACTAGTCGACATGAGTGTGCTCACTCAGGTCCTGG (GC)GTTG 3′ (SEQ ID NO: 24); Jones,S. T., and Bendig, M. M., Bio/Technology 9:88-89 (Erratum) (1991)) forcDNA amplification. For cloning of the heavy chain, mouse heavy chainvariable primers 1-12 were used as described (Jones, S. T., and Bendig,M. M., Bio/Technology 9:88-89 (Erratum) (1991)). All heavy chainsamplified best with primer 9; though lesser amplification was also seenwith primers 12, 10 and 6. The PCR products were isolated by low meltagarose fractionation and ligated into a vector. The light chain PCRproduct was ligated into PCR II vector (Invitrogen, San Diego, Calif.)The heavy chain PCR product from primer 9 was ligated into PCR II.1vector (Invitrogen, San Diego, Calif.). After transformation, theplasmid DNA was isolated and subjected to restriction digestion withEcoR1. Two clones from each heavy and light chain were expanded and theDNA harvested. Both strands of the cDNA clones were sequenced using T7and M13 primers with an ABI Prism automated sequencing apparatus. ThecDNA sequences and deduced amino acid sequences are shown in FIGS. 11-16(SEQ ID NOS: 4-15).

EXAMPLE 4 Preparation and Characterization of Chimeric and HumanizedAntibodies

[0175] In designing the sequence for a chimeric or humanized antibody,there are many parameters to consider. In the constant regions, a wholeantibody may be made, or an antibody fragment (Fab and Fab′2) can bemade. The constant regions may be murine or human. It is an acceptedpractice to replace murine constant regions with human constant regions,thus forming a “chimeric” antibody. Chimeric antibodies are lessimmunogenic than murine antibodies and are thus more acceptable in theclinic.

[0176] The subclass of the antibody must also be considered. It is mostcommon to express recombinant antibodies as IgGs, but within this class,one must choose amongst recombinant chimeric human IgG1, IgG2, IgG3, andIgG4. These subclasses have different biological properties. The presentinventors took a conservative approach of using IgG2 because 1) thestrong complement activating properties of IgG1 and IgG3 were not neededfor this antibody and 2) IgG2 may be more straightforward to manufacturethan IgG4. Any of the other subclasses could be made with the samespecificity following similar strategies.

[0177] There are also parameters to consider in designing the variableregion. The antibodies could be constructed to be chimeric or humanized.The chimeric antibody (murine V region, human constant region) is a moreconservative approach, and virtually guarantees very similarantigen-binding activity to the murine antibody. With humanization,there is the risk of reducing the affinity and/or biological activity ofthe antibody, but it can be presumed that the antibody will be lessimmunogenic. The present inventors have produced chimeric antibody aswell as three forms of the humanized antibody.

[0178] Depending upon the strategy taken, humanization of any particularantibody can result in many different variable regions. At the simplestlevel, humanization consists of choosing a human variable region toserve as a template, and then deciding which residues should be “human”and which “murine”. Thus, the choice of both the human template andwhich residues to maintain as human will affect the final sequence.

[0179] In general, the strategy the present inventors have taken is tochoose from among the human germline variable region genes for thetemplates. Alternatively, one can choose from rearranged variable regiongenes, both those which have and have not undergone somatic mutation.The rationale for the first strategy is that somatic mutations canintroduce immunogenic epitopes, while germline genes would have lesspotential for doing so. The selection was further limited to germlinegenes which are known to be rearranged and expressed as functionalproteins in humans.

[0180] The choice of which germline gene to use as template is governedby the overall sequence similarity between the murine sequence and thehuman sequence; the structural similarities between the two sequences(Chothia and Lesk, J. Mol. Biol. 196:901 (1987)); the anticipatedability of the chosen heavy chain template to pair with the chosen lightchain template; and the presence of the germline gene in the majority ofhumans. The choice of which residues should be murine is governed bywhich residues are thought to come in contact with antigen and which arenecessary to maintain the positioning and orientation of those residueswhich might contact antigen.

[0181] Variable regions were assembled from oligonucleotides andinserted into expression vectors containing the human gamma 2 constantregion (for the VH region) and human kappa constant region (for the VLregion). Heavy and light chain vectors were verified by nucleotidesequence and ability to direct the synthesis of antigen bindingimmunoglobulin (Ig) in COS cells (transient expression). Selected heavyand light chain vectors were then cotransfected into CHO cells toproduce stable cell lines expressing the chimeric and humanizedantibodies. Antibody was purified and tested for activity by antigenbinding ELISA, ability to block the inhibitory activity of α2-AP in aplasmin assay, and ability to facilitate lysis of human clots byurokinase.

[0182] A. Construction of Chimeric and Humanized Antibody Vectors

[0183] A functional light chain variable region is formed by therearrangement and juxtaposition of a V gene segment and J gene segment.Therefore, it was necessary to find the best match for each of thesesegments and combine them to form a human template. A FASTA search(using the Wisconsin Package Interface) of amino acids 1-95 (Kabatnumbering system; V gene proper) of murine 77A3 (m77A3) light chainagainst a database of human Vk germline genes showed that m77A3 isclearly most similar to the human VkI subgroup (69.2% −71.6% identity vsless than 60% identity to sequences outside this subgroup). From amongthe Vk I sequences, the sequence with GenBank accession # X59312 (alsoknown as the O2/O12 gene) was chosen as a likely candidate because ofthe match with structurally important positions and because of itsprevalent expression in humans. The human template for the light chainwas completed by the addition of the human Jk2 sequence. This J regionwas chosen because of its high degree of similarity with the murine Jregion of 77A3.

[0184] A functional heavy chain variable region is formed by therearrangement and juxtaposition of a V gene segment, a D gene segment,and a J gene segment. Therefore, it was necessary to find the best matchfor each of these segments and combine them to form a human template. AFASTA search (using the Wisconsin Package Interface) of amino acids 1-94(Kabat numbering system; V gene proper) of murine 77A3 heavy chainagainst a database of human VH germline genes showed that m77A3 isclearly most similar to the human VH7 family (77% identity) with thehuman VH1 family having the next best match (about 60% identity). Thehuman VH7 family is mostly composed of pseudogenes; the only active gene(7-04.1, Accession # X62110) is polymorphic in the human population(i.e. not all people have it) and therefore, in some people, this V genecould be more immunogenic than others. As an alternative human templatefor the heavy chain, the V gene with accession number Z12316 (1-18 gene)was chosen. This sequence is very similar to 7-04.1 except for the H2loop and FR3 region. A human template for the D region was notconsidered because this region lies entirely within the H3 loop, thesequence of which is generally pivotal for antigen binding and thereforelikely to entirely follow the murine sequence in a humanized antibody.The human template for the heavy chain was completed by the addition ofthe human JH5 sequence. This J region was chosen because of its highdegree of similarity with the murine J region of 77A3.

[0185] Following the selection of human templates for the heavy andlight chain variable regions, it was necessary to determine whichpositions should follow the murine sequence vs which positions shouldfollow the human sequence. The following criteria were used in selectingpositions to follow the murine sequence: all positions falling withinthe CDR loops; all positions known to influence the conformation and/orspatial position of CDR loops (so called structural determinants;Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Lesk and Tramontano, in:Antibody Engineering, W. H. Freeman and Co., pp.7-38 (1992)); residueswhich were close enough to interact with residues in the CDR loops; andresidues at or proximal to the VH-VL domain interface. All otherresidues followed the human sequence. These items are discussed ingreater detail below.

[0186] Positions falling within the CDR loops are shown enclosed withinthe boxes with solid borders and structural determinants are marked withan * in the row below the position number in FIGS. 17-19.

[0187] In order to determine which residues were close enough tointeract with the CDR loops, it was necessary to generate an approximatemolecular model of the Fv region of murine 77A3. The molecular model wasbuilt based on the combined variable light chain of an anti-lysozyme mAb(D1.3) and the variable heavy chain of an anti-neuraminidase mAb (lncca)as structural template. CDR loop sequences were assigned to canonicalloop conformations and a possible conformation for CDR H3 was extractedform the Protein Data Bank. The modeling building protocol followedprocedures described by Bajorath & Novotny (Therapeutic Immunol.2:95-105 (1995)). Likewise, residues at or proximal to the VH-VL domaininterface were identified and the murine residues were used for thehumanized antibody. In all, for h77A3-1 heavy chain, h77A3-2 heavychain, and for the common light chain there were 7, 18, and 11 murineresidues, respectively, used outside of the CDR loops.

[0188] In order to prepare vectors encoding these chains, the amino acidsequence must be back translated into nucleotide sequence. For the mostpart, this was done simply by using the nucleotide sequence from thehuman template in cases where the amino acid residue is derivedspecifically from the human template; otherwise, the nucleotides fromthe murine sequence were used. At a few positions, silent substitutionswere made in order to eliminate restriction sites.

[0189] Finally, signal peptides must be added to the sequence. For boththe chimeric and humanized light chains, signal peptides correspondingto that of the murine 77A3 light chain were used. For the chimeric andhumanized heavy chains, the same signal peptide as for the light chainswas used. Alternatively, signal peptides corresponding to that of murine77A3 VH or any other signal peptide can be used in the chimeric andhumanized heavy chains.

[0190] Two humanized antibodies were created: h77A3-1 and h77A3-2. Athird version of the humanized heavy chain was created by including anoligonucleotide designed for h77A3-1 in the construction of h77A3-2.This resulted in a hybrid molecule that was identical to h77A3-2 exceptfor amino acids Ser and Leu at positions 9 and 11 of the heavy chain.One chimeric antibody, c77A3, was generated.

[0191] Amino acid and nucleotide sequences of h77A3-1 and h77A3-2 heavyand light chains are shown in FIGS. 17-19 (SEQ ID NOS: 16-21). Thecommon light chain is shown in FIG. 17 (mature protein is amino acidresidues 1 to 107 of SEQ ID NO: 17). The heavy chain of h77A3-1 is shownin FIG. 18 (mature protein is amino acid residues 1 to 119 of SEQ ID NO:19). The heavy chain of h77A3-2 is shown in FIG. 19 (mature protein isamino acid residues 1 to 123 of SEQ ID NO: 21).

[0192] Expression vectors for chimeric and humanized 77A3 light andheavy chains were prepared in three stages: (1) construction ofcassettes containing human light or heavy chain constant region genes(pD16-hCka and pD20-hγ2a, respectively); (2) preparation of a PCRproduct containing the light or heavy chain variable region; and (3)insertion of the variable region into the appropriate expressioncassette.

[0193] Plasmid pD13 was constructed and derived from the pcDNA3 plasmid(Invitrogen) in two steps. The SV40 promoter/enhancer and neomycinresistance genes were removed from pcDNA3 by digestion with NaeI andisolation of the 3.82 kb fragment. These genes were replaced by the SV40promoter/enhancer and dhfr gene from pSV2-dhfr. The DNA containing thepSV2-dhfr sequence was isolated as a 1.93 kb fragment after digestionwith PvuII and BamHI. The 3.82 and 1.93 kb fragments were ligatedtogether and used to transform MC1061 bacteria following filling in theprotruding ends of the 1.93 kb fragment from pSV2-dhfr. The correctproduct (designated pD12) was confirmed by the release of an 890 bpfragment following HindIII digestion.

[0194] The polylinker was replaced with alternative restriction sites bydigesting the resultant vector above with Asp718 and Bsp120I. Thefollowing oligonucleotides were annealed to the vector and cloned byExoIII cloning (K. Hsiao, Nucl. Acid. Res. 21:5528-5529 (1993)) tocomplete the plasmid pD13:5′ TAGGGAGACCCAAGCTTGGTACCAATTTAAATTGATATCTCCTT AGGTCTCGAGTCTCTAGATAACCGGTCAATCGATTGGGATTCTT 3′ (SEQ ID NO:25) and5′    GACACTATAGAATAGGGCCCTTCCGCGGTTGGATCCAACACGTGAAGCTAGCAAGCGGCCGCAAGAATTCCAATCGATTGACCGGTTA 3′ (SEQ ID NO:26).

[0195] The resulting plasmid was used to transform competent E. coliDH5α and the correct product was confirmed by sequencing the polylinkerregion.

[0196] Plasmid pD16 was derived from the pcDNA3 plasmid (Invitrogen) ina series of steps which: add a polylinker sequence upstream of the CMVpromoter for linearization; delete the SV40 promoter/enhancer andneomycin resistance gene and replace them with the histone H3transcription termination sequence, the SV40 promoter (enhancer deleted)and DHFR gene; and insert the gastrin transcription termination sequenceupstream of the CMV promoter.

[0197] pcDNA3 (Invitrogen) was digested with BglII and annealed to thefollowing oligonucleotides: 5′ primer:5′-GATCTGCTAGCCCGGGTGACCTGAGGCGCGCCTTTG GCGCC-3′ (SEQ ID NO:27); and3′ primer: 3′-ACGATCGGGCCCACTGGACGCCGCGCGGAAACCGCG GCTAG-5′ (SEQ IDNO:28).

[0198] The plasmid was then ligated. After ligation, the resultingplasmid (pcDNA3-LSI) was used to transform competent E. coli DH5α andthe correct construct was confirmed by release of a 230 bp fragmentfollowing restriction enzyme digestion with NheI and NruI.

[0199] Plasmid pcDNA3-LSI was then digested with NgoMI, PvuI and BsmI.Following digestion, a 2.0 kb NgoMI-PvuI fragment was isolated. PlasmidpD12 (described above) was digested with PvuI and SphI to remove theSV40 enhancer and a 3.6 kb fragment was isolated. The followingoligonucleotides, encoding the histone H3 transcription terminationsequence were annealed and then ligated with the 2.0 kb NgoMI-PvuIfragment and 3.6 kb PvuI-SphI fragment: 5′ primer:5′-CCGGGCCTCTCAAAAAAGGGAAAAAAAGCATG-3′ (SEQ ID NO:29); and 3′ primer:3′-CGGAGAGTTTTTTCCCTTTTTTTC-5′ (SEQ ID NO:30).

[0200] The resulting plasmid pcTwD-LS1 was confirmed by the productionof 3.3, 0.95, 0.82 and 0.63 kb fragments after digestion with NheI plusNciI and the production of 4.2, 1.0, 0.26 and 0.23 kb fragments afterdigestion with SphI plus BstEII.

[0201] Insertion of the gastrin transcription termination sequence toform plasmid pD16 was accomplished by digesting pcTwD-LS1 with BssHIIand NarI and isolating the 5.7 kb fragment and ligating with thefollowing annealed oligonucleotides: 5′ primer:5′-CGCGCCGGCTTCGAATAGCCAGAGTAACCTTTTTTTTTAATTTTATTTTATTTTATTTTTGAGATGGAGTTTGG-3′ (SEQ ID NO:31); and 3′ primer:3′-GGCCGAAGCTTATCGGTCTCATTGGAAAAAAAAATT AAAATAAAATAAAATAAAAACTCTACCTCAAACCGC-5′ (SEQ ID NO:32).

[0202] After ligation, the product was used to transform competent E.coli MC1061 and the correct construction was confirmed by the productionof 4.8, 0.66 and 0.31 kb fragments after digestion with NgoMI plus SpeIand the production of 3.3, 1.0, 0.82 and 0.67 kb fragments followingdigestion with NgoMI plus NcoI.

[0203] Plasmid pD17 was derived from pD16 by the removal of the NheIsite from the linearization polylinker. This was accomplished bydigestion of pD16 with BstII and NheI and filling the protruding endsusing Klenow polymerase. The reaction mixture was self-ligated and usedto transform competent E. coli DH5α.

[0204] pD17 was digested with Asp718I and Bsp120I to remove a polylinkerwhich was replaced by the 113 bp Asp718I/Bsp120I polylinker from pD13.After ligation, the resulting intermediate plasmid pD20 had the NheIsite required for inserting heavy chain V genes. pD20 was distinguishedfrom pD17 by linearization with NheI, and distinguished from pD13 bylinearization with BssH II which cuts only once within the linearizationsite polylinkers of pD16, pD17 and pD20. Finally, DNA sequencing wasused to confirm the polylinker in pD20.

[0205] A 2.9 kb EcoRI fragment was isolated from pGk.11 (Walls et al.,Nucl. Acid. Res. 21:2921-2929 (1993)) and this was ligated into theplasmid pD13 (described above) previously digested with EcoRI. Thisconstruct (pD13-hCka) containing the human Cκ exon and flanking intronsequences was used to transform E. coli DH5α and the correct product wasconfirmed by restriction digestion. Digestion with EcoRI resulted infragments of 5.7, 2.8 and 0.3 kb and digestion with SacI resulted infragments of 7.1, 1.1 and 0.5 kb.

[0206] Construction of the light chain expression cassette was completedby removing the Cκ fragment along with the flanking polylinker sequencesfrom pD13 and inserting it into pD16. Plasmid pD13-hCka was digestedwith Asp718I and Bsp120I to release the Cκ fragment and polylinkersequences. The same enzymes were used to linearize pD16 and the Cκcontaining fragment was ligated into pD 16 to form pD16-hCka. Followingtransformation of DH5α E. coli and amplification, the correct constructwas confirmed by the release of 2.9 kb fragment following digestion withAsp718I and Bsp120I and linearization following digestion with arestriction enzyme present in pD16, but not pD13. The nucleotidesequence was also confirmed by sequencing various regions of theconstruct.

[0207] A genomic DNA fragment encoding the human γ2 gene waspreassembled in pIC, and then transferred into pD20 as follows. Phageclone Phage 5A (Ellison and Hood, Proc. Natl. Acad Sci, 79: 1984-1988(1982)), containing the human γ2 gene was digested with HindIII andcloned into the HindIII site of pUC18 to form the vector pγ2. In pγ2,the 5′ end of the γ2 gene is adjacent to the polylinker region.

[0208] pG was derived from pSV2-gpt by digestion with Hind III and BglII, Klenow fill in, and religation. This served to remove a 121 bp HindIII-Bgl II fragment. pγ2 was then digested with BamH I and inserted intothe BamH I site of pG to form pGγ2.2. pGγ2.2 contains a BglII site 3′ ofthe coding region that would interfere with later cloning steps. Toremove this restriction site, pGγ2.2 was first digested with Bgl II, thesticky ends filled in by Klenow DNA polymerase I, then the plasmidreligated. The resulting intermediate plasmid, pGγ2.3 was screened forlack of digestibility with Bgl II.

[0209] For purposes of later cloning in variable region genes, it wasimportant to provide a restriction site in the γ2 containing cassette.This is conveniently done by mutating the nucleotides encoding the firsttwo amino acids of the CH1 exon to encode an Nhe I site (Coloma M. J. etal., J. Immunological Methods 152:89-104(1992)). Previously, an Nhe I toBst E II fragment from the human γ4 gene was cloned. In this region,human γ2 and human γ4 genes encode identical amino acids. Thus, the γ4containing vector (pIChγ4.1) could serve as a source for the 5′ end ofthe γ2 gene. This vector was obtained as follows: The 8.6 kb BamH Ifragment from Phage 5D (Ellison, J. et al DNA 1:11-18 (1981)),containing the human γ4 gene, was subcloned into pUC, resulting in theplasmid pUChγ4. pUChγ4 served as the template for a PCR reactioninvolving the following primers: sense primer:5′-ATCGATGCTAGCACCAAGGGCCCA-3′ (SEQ ID NO: 33); and antisense primer:5′-CTCGAGGGGTCACCACGCTGCTGA-3′ (SEQ ID NO: 34). The sense primercontained a Cla1 site for subcloning the PCR product into pIC20R (MarshJ. L., et al., Gene 32: 481-485 (1984)) adjacent to a synthetic Nhe1site (underlined). Note that the bases for the Nhe1 site can encode thefirst two amino acids (Alanine and Serine) for the human γ1, γ2, γ3 orγ4 CH1 exon. The antisense primer has an Xho I site for subcloning intopIC20R, next to a BstE II site (underlined) which is in the CH1 exon ofthe human γ4 and γ2 gene. The PCR product formed was restricted with ClaI+Xho I then ligated into pIC20R which had been digested by the sameenzymes, to generate the intermediate pIChγ4. 1.

[0210] pGγ2.3 was digested with BamH I and HinD III and a 6.1 Kbfragment including the human γ2 gene locus was isolated from a 1.4%agarose gel for purification by the Qiaex™ gel extraction kit (Qiagen,Chatsworth, Calif.). The 2.9 Kb pIChγ4.1 plasmid was treated in asimilar manner, and the two fragments were ligated together to form theintermediate vector pIChγ2. 1. To screen, an EcoR I digest yieldedappropriate fragment sizes of 6.3 Kb and 2.6 Kb.

[0211] pIChγ2.1 contained a duplication of the 5′ portion of the humanγ2/γ4 CH1 exon. In order to remove the duplicated region, it wasdigested with BstE II giving fragment sizes of 4.0 Kb, 1.8 Kb, 1.6 Kb,1.1 Kb, and 0.4 Kb. The 4.0 Kb fragment was isolated from a 1.4% agarosegel, while the 1.6 Kb fragment was separated and isolated away from the1.8 Kb fragment in 4% NuSieve™ GTG (FMC Bioproducts, Rockland, Me.)agarose. Both fragments were purified by Qiagen gel extraction prior toligating them together to prepare pIChγ2.2. In order to confirm theproper orientation of the two fragments the following primers were usedto determine that the 3′ portion of the human γ4 CH1 exon's BstE IIsticky end had joined with the 5′ end of the human γ2 CH1 exon (thusforming a contiguous human γ2 locus in pIC20R): sense primer:5′-AACAGCTATGACCATGATTAC-3′ (SEQ ID NO:35); and antisense primer:5′-CACCCAGCCTGTGCCTGCCTG-3′ (SEQ ID NO:36).

[0212] The sense primer is homologous to sequence 5′ of the pIC20R EcoRI site that is adjacent to the Cla I site. The antisense primer waschosen to be 500 bp downstream of the sense strand primer, and ishomologous to sequence within the human γ2 CH1 to CH2 intron. Thus,visualization of a 500 bp PCR product in a 1.4% agarose gel confirmedthat the hybrid human γ4-γ2 CH1 exon formed and was oriented in acontiguous manner to the remainder of the locus. pIChγ2.2 was digestedwith EcoR I to give the expected 2.6 Kb and 1.9 Kb fragments. The entirehuman γ2 CH1 exon was confirmed by DNA sequencing.

[0213] The 1.8 Kb Nhe I+HinD III fragment containing the human γ2 genelocus was removed from pIChγ2.2 for ligation into plasmid pD20 opened byNhe I+HinD III. The resulting vector is the expression cassettepD20-hγ2a.

[0214] The variable region (V) genes for both chimeric and humanizedantibodies were synthesized by a modification of the non templatespecific PCR protocol (Prodromou C., and Pearl L. H., Protein Eng. 5:827-829 (1992)). The PCR products included DNA encoding both the signalpeptide and variable region proper as well as flanking sequences tofacilitate insertion into the vector as well as correct splicing (lightchain only).

[0215] The following primers were used:

[0216] LH1, sense chimeric 77A3 VH outer primer (30 mer),5′-CGATTGGAATTCTTG CGGCCGCTTGCTAGC-3′ (SEQ ID NO: 37);

[0217] LH2, sense chimeric 77A3 VH primer 1 (80 mer),5′-CTTGCGGCCGCTTGCTA GCATGGATTGGGTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAAGTATCCAAGCACAGA-3′ (SEQ ID NO: 38);

[0218] LH3, anti-sense chimeric 77A3 VH primer 2 (80 mer),5′-CTTGACTGTTTC TCCAGGCTTCTTCAGCTCAGGTCCAGACTGCACCAACTGGATCTGTGCTTGGATACTTTGGGCAGCTG-3′ (SEQ ID NO: 39);

[0219] LH4, sense chimeric 77A3 VH primer 3 (80 mer), 5′-CTGAAGAAGCCTGGAGAAACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCAC AAACTATGGAATGAACTGGGT-3′(SEQ ID NO: 40);

[0220] LH5, anti-sense chimeric 77A3 VH primer 4 (80 mer),5′-TCTTGGTGTTTAT CCAGCCCATCCACTTTAAACCCTTTCCTGGAGCCTGCTTCACCCAGTTCATTCCATAGTTTGTGAAG-3′ (SEQ ID NO: 41);

[0221] LH6, sense chimeric 77A3 VH primer 5 (80 mer), 5′-AGTGGATGGGCTGGATAAACACCAAGAGTGGAGAGCCAACATATGCTGAAGAGTTCAA GGGACGGTTTGCCTTCTCTTTG-3′(SEQ ID NO: 42);

[0222] LH7, anti-sense chimeric 77A3 VH primer 6 (80 mer),5′-TCCTCATTTTTGA GGTTCTTGATCTGCAAATTGGCAGTGCTGGCAGAGGTTTCCAAAGAGAAGGCAAACCGTCCCTTGAA-3′ (SEQ ID NO: 43);

[0223] LH8, sense chimeric 77A3 VH primer 7 (80 mer), 5′-GCAGATCAAGAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGATGGGTACCT GGGACCTATGCCATGGACT-3′(SEQ ID NO: 44);

[0224] LH9, anti-sense chimeric 77A3 VH primer 8 (80 mer),5′-TGGGCCCTTGGTGC TAGCTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATGGCATAGGTCCCAGGTACCC-3′ (SEQ ID NO: 45);

[0225] LH10, anti-sense murine 77A3 VH outer primer (29 mer),5′-GGGAAGACGGATG GGCCCTTGGTGCTAGC-3′ (SEQ ID NO: 46);

[0226] LH11, sense chimeric 77A3 VL outer primer (30 mer),5′-ATTTAAATTGAT ATCTCCTTAGGTCTCGAG-3′ (SEQ ID NO: 47);

[0227] LH12, sense chimeric 77A3 VL primer 1(79 mer),5′-ATTTAAATTGATATCTCC TTAGGTCTCGAGATGAGTGTGCTCACTCAGGTCCTGGCGTTGCTGCTGCTGTGGCTTACAG-3′ (SEQ ID NO: 48);

[0228] LH13, anti-sense chimeric 77A3 VL primer 2 (78 mer),5′-AGATGCAGATAGG GAGGCTGGAGACTGAGTCATCTGGATGTCACATCTGGCACCTGTAAGCCACAGCAGCAGCAACGC-3′ (SEQ ID NO: 49);

[0229] LH14, sense chimeric 77A3 VL primer 3 (78 mer),5′-GTCTCCAGCCTCCCTA TCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTA-3′ (SEQ ID NO: 50);

[0230] LH15, anti-sense chimeric 77A3 VL primer 4 (78 mer),5′-TATAGACCAG GAGCTGAGGAGATTTTCCCTGTTTCTGCTGATACCATGCTAAATAATTGTGAATATTCCCACTTGCTC-3′ (SEQ ID NO: 51);

[0231] LH16, sense chimeric 77A3 VL primer 5 (78 mer), 5-AAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGT TCAGTGGCAGTGGATCA-3′(SEQ ID NO: 52);

[0232] LH17, anti-sense chimeric 77A3 VL primer 6 (78 mer);5′-CTCCCAAAATCT TCAGGCTGCAGGCTGTTGATCCTGAGAGAAAATTGTGTTCCTGATCCACTGCCACTGAACCTTGAT-3′ (SEQ ID NO: 53);

[0233] LH18, sense chimeric 77A3 VL primer 7 (78 mer),5′-GCCTGCAGCCTGAAGATTTTGGGAGTCATTACTGTCAACATTTTTGGACCACTCCGTGGACGTTCGGTGGAGGCACCA-3′ (SEQ ID NO: 54);

[0234] LH19, anti-sense chimeric 77A3 VL primer 8 (81 mer),5′-TTCCAATCGATTGA CCGGTTATCTAGAGACTCGAGACTTACGTTTGATTTCCAGCTTGGTGCCTCCACCGAACGTCCACGG-3′ (SEQ ID NO: 55);

[0235] LH20, anti-sense chimeric 77A3 VL outer primer (30 mer),5′-TCGATTGA CCGGTTATCTAGAGACTCGAGA-3′ (SEQ ID NO: 56);

[0236] LH21, anti-sense humanized 77A3 VL primer 2 (78 mer),5′-AGATGCAGATA GGGAGGATGGAGACTGAGTCATCTGGATGTCACATCTGGCACCTGTAAGCCACAGCAGCAGCAACGC-3′ (SEQ ID NO: 69)

[0237] LH22, sense humanized 77A3 VL primer 3 (78 mer), 5′-GTCTCCATCCTCCCTATCTGCATCTGTGGGAGACAGAGTCACCATCACATGTCGAGCAAG TGGGAATATTCACAATTA-3′(SEQ ID NO: 70)

[0238] LH23, sense humanized 77A3 VL primer 5 (78 mer), 5′-AAATCTCCTCAACTCCTGGTCTATAATGCAAAAACCTTAGCAAGTGGTGTGCCATCAAG GTTCAGTGGCAGTGGATCA-3′(SEQ ID NO: 71)

[0239] LH24, anit-sense humanized 77A3 VL primer 6 (78 mer),5′-CTCCCAAAATC TTCAGGCTGCAGGCTGCTGATGGTGAGAGTAAAATCTGTTCCTGATCCACTGCCACTGAACCTTGAT-3′ (SEQ ID NO: 72)

[0240] LH25, sense humanized 77A3 VH-1 primer 1 (80 mer),5′-CTTGCGGCCGCTTG CTAGCATGAGTGTGCTCACTCAGGTCCTGGCGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTC-3′ (SEQ ID NO: 57);

[0241] LH26, anti-sense humanized 77A3 VH-1 primer 2 (80 mer);5′-GACTGAGGCT CCAGGCTTCTTCAGCTCAGATCCAGACTGCACCAACTGGATCTGACATCTGGCACCTGTAAGCCACAGCA-3′ (SEQ ID NO: 58);

[0242] LH27, sense humanized 77A3 VH-1 primer 3 (80 mer),5′-GAGCTGAAGAAGC CTGGAGCCTCAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTG-3′ (SEQ ID NO: 59);

[0243] LH28, anti-sense humanized 77A3 VH-1 primer 4 (80 mer)5′-TGGTGTTTATC CAGCCCATCCACTCTAAACCTTGTCCTGGAGCCTGTCGCACCCAGTTCATTCCATAGTTTGTGAAGGTA-3′ (SEQ ID NO: 60);

[0244] LH29, sense humanized 77A3 VH-1 primer 5 (80 mer),5′-TAGAGTGGATGGG CTGGATAAACACCAAGAGTGGAGAGCCAACATATGCTGAAGAGTTCAAGGGACGGTTTGTCTTCTCT-3′ (SEQ ID NO: 61);

[0245] LH30, anti-sense humanized 77A3 VH-1 primer 6 (80 mer),5′-TCAGCTTTGAGG CTGCTGATCTGCAAATAGGCAGTGCTGACAGAGGTGTCCAAAGAGAA GACAAACCGTCCCTTGAACTC-3′ (SEQ ID NO: 62);

[0246] LH31, sense humanized 77A3 VH-1 primer 7 (80 mer),5′-TTTGCAGATCAG CAGCCTCAAAGCTGAGGACACGGCTGTGTATTTCTGTGCAAGATGGGTACCTGGGACCTATGCCATGG-3′ (SEQ ID NO: 63);

[0247] LH32, anti-sense humanized 77A3 VH-1 primer 8 (80 mer),5′-GCCCTTGGTG CTAGCTGAGGAGACGGTGACCGTGGTTCCTTGACCCCAGTAGTCCATGGCATAGGTCCCAGGTACCCATC-3′ (SEQ ID NO: 64);

[0248] LH33, anti-sense humanized 77A3 VH-2 primer 2 (80 mer),5′-TGCTGTGGCT TACAGGTGCCAGATGTCAGATCCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCCTCAGTC-3′ (SEQ ID NO: 65);

[0249] LH34, sense humanized 77A3 VH-2 primer 5 (80 mer),5′-TAGAGTGGATGGGC TGGATAAACACCAAGAGTGGAGAGCCAACATATGCTGAAGAGTTCAAGGGACGGTTTACCTTCACC-3′ (SEQ ID NO: 66);

[0250] LH35, anti-sense humanized 77A3 VH-2 primer 6 (80 mer),5′-TCAGATCTGAG GCTCCTGATCTCCAAATAGGCAGTGCTCGTAGAGGTGTCCAAGGTGAAGGTAAACCGTCCCTTGAACTC-3′ (SEQ ID NO: 67); and

[0251] LH36, sense humanized 77A3 VH-2 primer 7 (80 mer, 5′-TTTGGAGATCAGGAGCCTCAGATCTGACGACACGGCTGTGTATTTCTGTGCAAGATGGGTACCTGGGACCTATGCCATGG-3′ (SEQ ID NO: 68).

[0252] Table 3 summarizes how the above primers were used in thenon-template PCR protocol. TABLE 3 Use of primers in non-template PCRHeavy chains Light chains chimeric humanized-1 humanized-2 chimerichumanized outer primer LH1 (SEQ LH1 (SEQ ID LH1 (SEQ ID LH11 (SEQ LH11(SEQ (sense) ID NO:37) NO:37) NO:37) ID NO:47) ID NO:47) V1 (sense) LH2(SEQ LH25 (SEQ LH25 (SEQ LH12 (SEQ LH12 (SEQ ID NO:38) ID NO:57) IDNO:57) ID NO:48) ID NO:48) V2 LH3 (SEQ LH26 (SEQ LH33 (SEQ LH13 (SEQLH21 (SEQ (antisense) ID NO:39) ID NO:58) ID NO:65) ID NO:49) ID NO:69)V3 (sense) LH4 (SEQ LH27 (SEQ LH27 (SEQ LH14 (SEQ LH22 (SEQ ID NO:40) IDNO:59) ID NO:59) ID NO:50) ID NO:70) V4 LH5 (SEQ LH28 (SEQ LH28 (SEQLH15 (SEQ LH15 (SEQ (antisense) ID NO:41) ID NO:60) ID NO:60) ID NO:51)ID NO:51) V5 (sense) LH6 (SEQ LH29 (SEQ LH34 (SEQ LH16 (SEQ LH23 (SEQ IDNO:42) ID NO:61) ID NO:66) ID NO:52) ID NO:71) V6 LH7 (SEQ LH30 (SEQLH35 (SEQ LH17 (SEQ LH24 (SEQ (antisense) ID NO:43) ID NO:62) ID NO:67)ID NO:53) ID NO:72) V7 (sense) LH8 (SEQ LH31 (SEQ LH36 (SEQ LH18 (SEQLH18 (SEQ ID NO:44) ID NO:63) ID NO:68) ID NO:54) ID NO:54) V8 LH9 (SEQLH32 (SEQ LH32 (SEQ LH19 (SEQ LH19 (SEQ (antisense) ID NO:45) ID NO:64)ID NO:64) ID NO:55) ID NO:55) outer primer LH10 (SEQ LH10 (SEQ LH10 (SEQLH20 (SEQ LH20 (SEQ (antisense) ID NO:46) ID NO:46) ID NO:46) ID NO:56)ID NO:56)

[0253] Briefly, 8 adjacent oligonucleotides which represent a syntheticlight or heavy chain V gene are synthesized (V1-V8 in Table 3). Foursense strand oligonucleotides alternate with 4 overlapping antisensestrand oligonucleotides of 78-81 nt in length. These PCR primers overlapeach other by 24-27 nt. Note that for oligonucleotide primers V1 and V8(Table 3), their 5′ end is designed to overlap with 15-30 nt of thevector sequence, while their 3′ end overlaps 48-65 nt of the signalpeptide (VI) or the V gene sequence (V8). The 8 oligonucleotide primersare all included in the same first round PCR. Reaction conditions forthis 1st round PCR were 0.125 picomoles of each primer, 10 μl of 10×Pfubuffer (Stratagene Inc., San Diego, Calif.), 10 nanomoles dNTP's(Boehringer Mannheim, Indianapolis, Ind.), 10% dimethylsulfoxide (DMSO),and 2.5 units cloned Pfu DNA polymerase I (Stratagene Inc., San Diego,Calif.) in a 100 μl reaction volume. Reactants were first denatured at95° C. for 5 min. annealed at 45° C. for 5 min, and extended at 72° C.for 1 min, followed by 25 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec, and extension at 72° C. for 30 sec. The25 cycles were followed by a final extension at 72° C. for 7 min in aPerkin-Elmer DNA Thermal Cycler (Norwalk, Conn.).

[0254] The amplified PCR product was electrophoresed through a 1.4%agarose gel and the smear of DNA running between approximately 350bp-500 bp was cut out prior to purification by the Qiaex™ II gelextraction kit (Qiagen, Chatsworth, Calif.). This purified non templatespecific PCR product served as the template for a 2nd round PCR. Tocomplete the 2nd round PCR, two additional outer primers are utilized.These outer primers are homologous to 29-30 nt of the vector sequencethat is either 5′ (sense primer) or 3′ (antisense primer) of thelinearized cloning site within the mammalian expression cassette vector.This allowed for the amplified PCR product to be subcloned into thevector by bacterial homologous recombination (Jones, D. H. and Howard,B. H., BioTechniques 10: 62-66 (1991)). Thus, the reaction conditionsfor the 2nd round PCR were 0.125 picomoles each of outer sense andantisense primers, 10 μl of 10×Pfu buffer, 10 nanomoles dNTP's, 10%DMSO, 2.5 units Pfu DNA polymerase I, and approximately 100 ng of 1stround PCR template DNA. The reactants underwent the same thermocycleprogram described above. Subsequently, the amplicand from this reactionwas removed from a 1.4% agarose gel and purified with the Qiagen™ II gelextraction kit.

[0255] 200 ng-1000 ng of PCR product was mixed with an equal weight oflinearized vector, and this mixture was used to transform 200 ml ofcompetent E. coli DH5α cells (GIBCO BRL/Life Technologies, Gaithersburg,Md.). Transformed cells were selected by 100 μg/ml ampicillin in LBagarose. Typically, pD16-hCka digested with Xho I was used forsubcloning light chain V genes. pD20-hg2a digested with Nhe I served asthe vehicle for heavy chain V gene constructs.

[0256] In order to confirm that the V gene of interest had been insertedinto the expression vector, two screens were performed. The primaryscreen was by PCR, while the secondary screen was by restriction digest.Each individual colony of bacteria was picked into 5 ml of T broth(GIBCO BRL/Life Technologies, Gaithersburg, Md.) containing 100 μg/mlampicillin and grown 8-16 hr at 37° C. with shaking. The conditions forthe PCR screen were 0.125 picomoles of both outer primers (Table 3), 2ml 10×Mg⁺² buffer (Boehringer Mannheim, Indianapolis, Ind.), 10nanomoles dNTP's (Boehringer Mannheim, Indianapolis, Ind.), 1 unit TaqDNA polymerase I (Boehringer Mannheim, Indianapolis, Ind.), and 1 μl ofthe liquid culture growth (which served as the source of DNA templatesince the cells lysed at high temperature) in a 20 μl volume. Reactantsfirst underwent denaturation at 94° C. for 5 min, followed by 25 cyclesof denaturation at 94° C. for 25 sec, annealing at 45° C. for 25 sec,and extension at 72° C. for 12 sec. The cycles were followed by a finalextension at 72° C. for 7 min. Positives were determined by sizecomparison relative to a DNA standard marker after electrophoresisthrough a 1.4% agarose gel.

[0257] For the secondary screen, midi DNA preparations (Qiagen,Chatsworth, Calif.) were made from bacterial pellets and a portion wasdigested with either Xho I (VL genes) or Nhe I (VH genes). Again, afterelectrophoresis through a 1.4% agarose gel, size comparison of thefragment released due to enzyme digestion served to identify potentiallypositive clones.

[0258] The above procedures were used to confirm the presence of apotentially correct insert. However, they were not specific enough todetect small errors in the sequence (insertions, deletions andsubstitutions). To determine which clones contained DNA encodingcomplete Ig genes, each potentially positive heavy chain clone wascotransfected into COS cells with each potentially positive light chainclone. Culture supernatants were screened by ELISA for the presence ofhuman IgG, and then for the presence of IgG binding to α2-antiplasmin(see below).

[0259] DNA for COS transfections was derived from midi DNA preparationsdescribed above. COS tranfections were performed in 60 mm dishes.Complete details of the DEAE—dextran technique employed have beendescribed (Linsley P. S. et al., J. Exp. Med. 173: 721-730 (1991)).Typically, 1.5 μg-6 μg of whole antibody is derived from small scale COStransfections

[0260] As a final confirmation, the V region inserts from the aboveclones were sequenced by the dideoxy nucleotide procedure.

[0261] B. Production of Humanized and Chimeric Antibodies

[0262] Once heavy and light chain vectors encoding each of the desiredantibodies were qualified, sufficient quantities of chimeric andhumanized antibody for testing in functional assays were needed. Thiswas first done as a scale-up of the COS transfections using the selectedvectors. Finally, stable cell lines were prepared by high copy numberelectroporation. The electroporation protocol of Barsoum (Barsoum, DNAand Cell Biology 9:293-300 (1990)) was followed with the exception that100 μg each of the heavy and light chain vector were used (followingrestriction with BssHII) and the electroporation was performed in PFCHOmedia (PX-CELL PFCHO media, JRH Biosciences, Lenexa, Kans.).

[0263] Transfected cells were selected in media containing either 20 nMor 100 nM methotrexate (MTX). Culture supernatants were assayed for thepresence of whole antibody using the non-specific IgG ELISA. Cells frommaster wells containing the most antibody in the supernatant wereexpanded into larger volumes. In some cases, the methotrexateconcentration was also increased in order to amplify the vector in thecell lines. The vector pairs in Table 4 were electroporated into DG44CHO cells. TABLE 4 Vector pairs for production of antibody Heavy ChainLight Chain Product Vector Vector c77A3 (chimeric 77A3) pD20-cR1.H1pD16-cR1.L1 h77A3-1 (humanized 77A3) pD20-hR1.H1 pD16-hR1.L1 h77A3-2(humanized 77A3) pD20-hR2.H1 pD16-hR1.L1 h77A3-3 (humanized 77A3)pD20-hR3.H1 pD16-hR1.L1

[0264] C. Purification of Humanized and Chimeric Antibodies

[0265] The purification of the antibody was first performed usingprotein-A affinity chromatography. A Pharmacia column, sized so that 5mg of antibody to be loaded per 1 ml of resin, was packed withPerseptive Biosystems Poros 50 A protein-A resin. The column was thensanitized according to the methods recommended by the resin supplier.The column was equilibrated with pyrogen free 10 mM sodium phosphate,150 mM sodium chloride pH 7.0 (PBS). The cell culture supernatant wasadjusted to pH of 7.0-7.5 and loaded on the column at a flow rate equalto 2-3 column volume/min (CV/min). The column was then washed with 15 CVpyrogen free PBS or until a stable base line has been achieved. Theantibody was eluted with 20 mM glycine/HCl pH 3.0 elution buffer. Theeluted peak was collected in a pyrogen free vessel that contained 1/20CV of 1 M Tris base solution. The pH of the eluted antibody solution wasadjusted to pH 8.0 with 1M Tris base immediately. The column was thencleaned with 5 CV 12 mM HCl solution. The column was stored in 20%ethanol/water at 4.0° C.

[0266] The antibody was next purified using anion exchangechromatography. A Pharmacia column, sized so that 5-10 mg of antibody tobe loaded per 1 ml of resin, was packed with Perseptive Biosystems PorosHQ 50 anion exchange resin. The column was then sanitized according tothe methods recommended by the resin supplier. The column wasequilibrated with pyrogen free 50 mM Tris/HCl, 50 mM NaCl, pH 8.0. Theprotein-A purified antibody adjusted to pH of 8.0 was loaded on thecolumn with flow rate equal to 1 CV/min. The column was then washed with5 CV pyrogen free 50 mM Tris/HCl, 1M NaCl pH 8.0. The antibody does notbind to this column under the running conditions and was present in theflowthrough fraction. The column was stored in 20% ethanol/water at 4.0°C. The antibody was then concentrated and diafiltered against PBS usinga 30K cut off membrane.

[0267] D. Non-Specific IgG ELISA to Detect Presence of Antibody

[0268] This ELISA detects whole antibody (containing both heavy andlight chain) and relies on a capture antibody specific for human IgG Fcregion and a conjugate specific for human kappa chains. In this assay,Immunlon II flat bottom plates (Dynatech) were coated with goatanti-human IgG (Fc specific, adsorbed on mouse IgG) (Caltag, Inc.catalog #H10000) at 0.5 μg/ml in carb/bicarb buffer pH 9.6 and thenblocked with PTB (PBS containing 0.05% Tween 20 and 1.0% BSA). Samplewas added (either undiluted or diluted in PTB or Genetic Systemsspecimen diluent), the plates were incubated o/n at 4° C. or for a fewhours at room temperature. After washing, conjugate (goat anti-humankappa conjugated with horseradish peroxidase from Southern Biotech) wasadded at 1:10000 in PTB. After approximately 1 hour incubation at roomtemperature, plates were washed and 100 μl chromagen/substrate was added(Genetic Systems chromagen diluted 1:100 into Genetic Systemssubstrate). After sufficient color development (usually 5 to 15 minutes)100 μl 1 N H₂SO₄ was added to stop the reaction. Optical densities weredetermined using a Biotek plate reader set at 450 and 630 nmwavelengths.

[0269] In the occasional case that none of the samples from small COStransfections showed the presence of whole antibody, similar ELISAs wereperformed to determine whether any light chain was being secreted. Inthis case, the plates were coated with a goat anti-human kappa chain at1 μg/ml. The rest of the assay was done exactly as above.

[0270] The assay was used for three purposes. First, to screen small COStransfections that were set up to qualify various heavy and light chainvectors. In this case, the presence or absence of a signal wassufficient and it was not necessary to quantify the amount of antibodypresent. Second, to determine which of many master wells from CHOtransfections were producing the most antibody. In this case, culturesupernatants were diluted so that relative signals could be compared andthe master wells containing the most antibody could be distinguished andthus selected for cloning and expansion. Thirdly, to determine amountsof antibody, either in culture supernatants or following purification.In this case, a standard consisting of either a chimeric or human IgG1or a human myeloma IgG2 were used. Both standard and sample wereserially diluted (2×) across a plate and sample concentration relativeto standard was determined by comparing position of the curves. Theconcentrations thus determined were used for following antibodyproduction during the cloning and amplification process and fordetermining specific activity in the antigen binding ELISA and any ofthe functional assays.

[0271] E. ELISAs to Show that Antibody is Capable of Binding to Antigen

[0272] This ELISA relies on an antigen capture and a human kappa chainspecific conjugate. It was used for two purposes. Initially, to qualifya vector, supernatants from COS transfections were screened for theability of antibody to bind to antigen. Vectors passing this test werethen submitted to DNA sequencing. Secondly, to determine relativeantigen binding ability of the various chimeric and humanizedantibodies. This ELISA is very similar to the non-specific IgG ELISAdescribed above except that the plates were coated with α2-antiplasmin(obtained from American Diagnostica) at 1 μg/ml in PBS.

[0273] To determine relative antigen binding ability of variousantibodies, scatter plots were used with log antibody concentrationalong the X axis and optical density along the Y axis. Antigenconcentration was determined either from the non-specific ELISA or basedon optical density of purified preparations. All three forms ofhumanized antibody (h77A3-1, -2, and -3) show antigen binding similar tothat of the chimeric antibody. Comparisons were not made with the murineantibody (m77A3) because the m77A3 cannot be detected in the assay asdescribed (the antibody-conjugate used in the second step recognizesonly human constant regions).

[0274] F. Functional Assays

[0275] Two functional assays were performed. The first, known as the“plasmin assay with chromogenic substrate” is based on the ability ofplasmin to convert Spectrozyme PL, H-D-Nle-HHT-Lys-pNA.2AcOH into pNA,which absorbs light at 405 nm. If unblocked α2-antiplasmin is present,little or no conversion occurs. Active antibody is capable of blockingthe inhibitory activity of α2-antiplasmin. The second assay, the clotlysis assay, is a measure of the ability of antibody along withurokinase to lyse preformed clots.

[0276] The plasmin assay with chromogenic substrate is designed based onthe action of plasmin on its chromogenic substrate according to thereaction:

[0277] The generation of pNA was monitored by the increase in absorptionat 405 nm using a SpectraMax 250 spectrophotometer. The addition ofα2-antiplasmin inhibits the plasmin activity and no increase inabsorption at 405 nm will be observed. Premixing of α2-antiplasmin withfunctional antibody blocks the ability of α2-antiplasmin to inhibit theplasmin activity. Plasmin activity was measured as the initial rate ofcolor development.

[0278] Assays are performed in 96 well microtiter plates. Thechromogenic substrate Spectrozyme PL, H-D-Nle-HHT-Lys-pNA.2AcOH, humanplasmin, and human α2-antiplasmin were purchased from AmericanDiagnostica. Stock and working solutions are prepared as follows:Spectrozyme PL stock solution—10 mM in H₂O; Spectrozyme PL workingsolution—1:12.5 dilution of stock solution in H₂O; human plasmin stocksolution—0.2 mg/ml in 50% glycerol, 50% 2 mM HCl; human plasmin workingsolution—1:12.5 dilution of stock solution in 0.11 mM HCl, which must beprepared immediately before use; human α2-antiplasmin stock solution—0.2mg/ml in PBS; and human α2-antiplasmin working solution—1:15 dilution ofstock solution in PBS. Stock solutions were stored at −70 and should notbe refrozen after thawing.

[0279] Reagents are added in the following order, with mixing after eachaddition: 80 ul antibody or PBS, 40 ul α2-antiplasmin working solution,40 ul plasmin working solution, and 40 ul Spectrazyme PL workingsolution. R is the rate of color development. Rp, which representsmaximum plasmin activity, is determined in wells lacking both antibodyand α2-antiplasmin. Ro, which represents minimal plasmin activity, isdetermined in wells lacking antibody. Rs is the rate of colordevelopment in the sample. Antibody activity is calculated as(Rs−Ro)/(Rp−Ro) * 100. Values should range between 0% and 100%. Antibodyactivity was plotted vs. amount antibody (on a log scale). Curvesgenerated by test antibody and standard (usually murine 77A3) werecompared.

[0280] The data for murine 77A3, c77A3, and h77A3-1 are shown in FIG.20. The curves for murine and chimeric 77A3 were superimposable. Thecurve for h77A3-2 indicates a potential small loss in activity (20-30%).

[0281] The clot lysis assays were performed as follows. Test clots wereformed in 96-well Corning #25805 microtiter plates by mixing 25 uL 16 mMCaCl₂, 50 uL of pooled human plasma, and 25 uL of 4 NIH unit/ml of humanalpha-thrombin (Sigma) in 30 mM Hepes buffer, pH 7.40. Plates wereincubated overnight at room temperature to allow clots to achievemaximum clot turbidities. Clot lysis was initiated by adding 10 uL ofantibody to give 5 or 10 ug/well and 100 uL of urokinase to give 1, 3 or5 units of urokinase/well (Abbott Labs) at pH 7.40. Plates were mixed ona table top microplate vortexer for 30 sec before the initial reading at405 nm to get values corresponding to 0% lysis. Plates were sealed withCorning sealing tape #430454 and incubated at 37° C. During the courseof 24 hrs, the decrease of turbidity was measured at 405 nm to quantifythe progress of clot lysis.

[0282] The results of a clot lysis experiment of humanized 77A3-1indicate that h77A3-1 enhances clot lysis dramatically in comparison tobuffer controls in each of the conditions tested. There was significantseparation between the humanized and murine 77A3 in clots containing 5ug antibody in the presence of 1 or 3 units of urokinase indicating thathumanized 77A3 was somewhat less active than murine 77A3, even thoughthe lysis profiles were similar at the remaining four conditions tested.It should be noted that murine RWR, a monoclonal antibody with a 10-foldlower affinity than murine 77A3, causes no lysis at 10 ug per clot inthe presence of 1 unit of urokinase and would give a lysis profile likebuffer control.

EXAMPLE 5

[0283] Preparation and Characterization of Single Chain Fv Fragments

[0284] A. Design and Expression of sFv Form of 77A3

[0285] The sFv fragment of an antibody is most commonly obtained by thetandem expression of the variable region of the antibody heavy chainalong with the variable region of the antibody light chain spaced by alinker of 15-20 amino acids. sFv fragments are expected to have superiorclot penetration to parent antibodies. Two constructs, p53-6 and p52-12,were prepared using murine variable regions with a VH-(linker)-VLpolarity using YPRSIYIRRRHPSPSLTT (SEQ ID NO: 73) as linker 1 forsFv77A3-1 and GGSGSGGSGSGGSGS (SEQ ID NO: 74) as linker 2 for sFv77A3-2.Both constructs were cloned into the pET-22b vector from Novagen andtransformed into the BL21 (DE3) strain of E. coli grown in minimal M9media. Though the majority of the His-tagged product was found ininclusion bodies, supernatants of cell lysate contained sufficientquantities of soluble sFv fragments for nickel-column purification.

[0286] sFv77A3-2 present in fractions 7-11 collected from anickel-column gave a single Coomasie staining band with a MW about30,000 agreeing well with the calculated MW of 29,986. A similar butmore weakly staining gel was obtained for sFv77A3-1.

[0287] B. Activity of sFv77A3-1 and sFv77A3-2

[0288] Preparations of both sFv77A3-1 and sFv77A3-2 were tested foralpha2-antiplasmin binding activity in a competition binding assay.Microplate wells coated with 77A3 were treated with mixtures ofbiotinylated-human alpha2-antiplasmin and either sFv77A3-1 or sFv77A3-2along with positive control 77A3 and negative control, mAb-59D8.Increasing quantities of 77A3 prevented binding of biotinylated-humanalpha2-antiplasmin whereas negative control 59D8 had little effect as ancompetitive inhibitor. With concentrations of test samples estimated byintensity of Coomasie stained bands, both sFv77A3-1 and sFv77A3-2completely inhibited the binding of biotinylated-human α2-antiplasminwith a profile of inhibition nearly superimposible to the parental 77A3reference.

[0289] Idiotypic markers present on 77A3 were probed with a sandwichELISA using a biotinylated polyclonal reagent rendered specific bymultiple immunoadsorbtion steps through columns bearing immunoglobulinsfrom man, mouse, baboon and cynomologous monkey (P Stenzel-Johnson & DYelton, Seattle). Microplate wells were coated with 77A3 and 59D8 ascontrols along with sFv77A3-1 and sFv77A3-2. It is evident that 77A3control and both sFv fragments bear idiotypic markers at each dosetested indicating that the sFv fragments “look” like the parental 77A3.

[0290] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0291] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0292] The disclosure of all references, patent application, and patentsreferred to herein are hereby incorporated by reference.

1 81 1 15 PRT Artificial Sequence Alpha-2 Antiplasmin Antibody 1 Xaa IleGln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val 1 5 10 15 2 5 PRTArtificial Sequence Alpha-2 Antiplasmin Antibody 2 Asp Ile Gln Met Thr 15 3 15 PRT Artificial Sequence Alpha-2 Antiplasmin Antibody 3 Xaa IleGln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val 1 5 10 15 4 381 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 4 atg agt gtg ctc actcag gtc ctg gsg ttg ctg ctg ctg tgg ctt aca 48 Met Ser Val Leu Thr GlnVal Leu Xaa Leu Leu Leu Leu Trp Leu Thr -20 -15 -10 -5 ggt gcc aga tgtgac atc cag atg act cag tct cca gcc tcc cta tct 96 Gly Ala Arg Cys AspIle Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 1 5 10 gca tct gtg gga gaaact gtc acc atc aca tgt cga gca agt ggg aat 144 Ala Ser Val Gly Glu ThrVal Thr Ile Thr Cys Arg Ala Ser Gly Asn 15 20 25 att cac aat tat tta gcatgg tat cag cag aaa cag gga aaa tct cct 192 Ile His Asn Tyr Leu Ala TrpTyr Gln Gln Lys Gln Gly Lys Ser Pro 30 35 40 cag ctc ctg gtc tat aat gcaaaa acc tta gca gat ggt gtg cca tca 240 Gln Leu Leu Val Tyr Asn Ala LysThr Leu Ala Asp Gly Val Pro Ser 45 50 55 60 agg ttc agt ggc agt gga tcagga aca caa ttt tct ctc agg atc aac 288 Arg Phe Ser Gly Ser Gly Ser GlyThr Gln Phe Ser Leu Arg Ile Asn 65 70 75 agc ctg cag cct gaa gat ttt gggagt cat tac tgt caa cat ttt tgg 336 Ser Leu Gln Pro Glu Asp Phe Gly SerHis Tyr Cys Gln His Phe Trp 80 85 90 acc act ccg tgg acg ttc ggt gga ggcacc aag ctg gaa atc aaa 381 Thr Thr Pro Trp Thr Phe Gly Gly Gly Thr LysLeu Glu Ile Lys 95 100 105 5 127 PRT Artificial Sequence Alpha-2Antiplasmin Antibody 5 Met Ser Val Leu Thr Gln Val Leu Xaa Leu Leu LeuLeu Trp Leu Thr -20 -15 -10 -5 Gly Ala Arg Cys Asp Ile Gln Met Thr GlnSer Pro Ala Ser Leu Ser 1 5 10 Ala Ser Val Gly Glu Thr Val Thr Ile ThrCys Arg Ala Ser Gly Asn 15 20 25 Ile His Asn Tyr Leu Ala Trp Tyr Gln GlnLys Gln Gly Lys Ser Pro 30 35 40 Gln Leu Leu Val Tyr Asn Ala Lys Thr LeuAla Asp Gly Val Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly ThrGln Phe Ser Leu Arg Ile Asn 65 70 75 Ser Leu Gln Pro Glu Asp Phe Gly SerHis Tyr Cys Gln His Phe Trp 80 85 90 Thr Thr Pro Trp Thr Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 95 100 105 6 381 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 6 atg agt gtg ctc act cag gtc ctg ggg ttg ctg ctgctg tgg ctt aca 48 Met Ser Val Leu Thr Gln Val Leu Gly Leu Leu Leu LeuTrp Leu Thr -20 -15 -10 -5 ggt gcc aga tgt gac atc cag atg act cag tctcca gcc tcc cta tct 96 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser ProAla Ser Leu Ser 1 5 10 gca tct gtg gga gaa act gtc acc gtc aca tgt cgagca agt ggg aat 144 Ala Ser Val Gly Glu Thr Val Thr Val Thr Cys Arg AlaSer Gly Asn 15 20 25 att cac aat tat tta gca tgg tat cag cag aaa cag ggaaaa tct cct 192 Ile His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly LysSer Pro 30 35 40 cag ctc ctg gtc tat aat gca aga acc tta gca gat ggt gtgcca tca 240 Gln Leu Leu Val Tyr Asn Ala Arg Thr Leu Ala Asp Gly Val ProSer 45 50 55 60 agg ttc agt ggc agt gga tca gga aca caa tat tct ctc aagatc aac 288 Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys IleAsn 65 70 75 agc ctg cag cct gaa gat ttt ggg agt tat tac tgt caa cat ttttgg 336 Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp80 85 90 agt aat ccg tgg acg ttc ggt gga ggc acc aag ctg gaa atc aaa 381Ser Asn Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 95 100 105 7127 PRT Artificial Sequence Alpha-2 Antiplasmin Antibody 7 Met Ser ValLeu Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr -20 -15 -10 -5 GlyAla Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 1 5 10 AlaSer Val Gly Glu Thr Val Thr Val Thr Cys Arg Ala Ser Gly Asn 15 20 25 IleHis Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 30 35 40 GlnLeu Leu Val Tyr Asn Ala Arg Thr Leu Ala Asp Gly Val Pro Ser 45 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn 65 70 75Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp 80 85 90Ser Asn Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 95 100 105 8381 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 8 atg agt gtgctc act cag gtc ctg gcg ttg ctg ctg ctg tgg ctt aca 48 Met Ser Val LeuThr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr -20 -15 -10 -5 ggt gccaga tgt gac atc cag atg act cag tct cca gcc tcc cta tct 96 Gly Ala ArgCys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 1 5 10 gca tct gtggga gaa act gtc acc atc aca tgt cga gca agt ggg aat 144 Ala Ser Val GlyGlu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn 15 20 25 att cac aat tattta gca tgg tat cag cag aaa cag gga aaa tct cct 192 Ile His Asn Tyr LeuAla Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 30 35 40 caa ctc ctg gtc tataat gca aaa acc tta gca gat ggt gtg cca tca 240 Gln Leu Leu Val Tyr AsnAla Lys Thr Leu Ala Asp Gly Val Pro Ser 45 50 55 60 agg ttc agt ggc agtgga tca gga aca caa ttt tct ctc aag atc aac 288 Arg Phe Ser Gly Ser GlySer Gly Thr Gln Phe Ser Leu Lys Ile Asn 65 70 75 agc ctg cag cct gaa gatttt ggg agt cat tac tgt caa cat ttt tgg 336 Ser Leu Gln Pro Glu Asp PheGly Ser His Tyr Cys Gln His Phe Trp 80 85 90 acc act ccg tgg acg ttc ggtgga ggc acc aag ctg gaa atc aaa 381 Thr Thr Pro Trp Thr Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 95 100 105 9 127 PRT Artificial Sequence Alpha-2Antiplasmin Antibody 9 Met Ser Val Leu Thr Gln Val Leu Ala Leu Leu LeuLeu Trp Leu Thr -20 -15 -10 -5 Gly Ala Arg Cys Asp Ile Gln Met Thr GlnSer Pro Ala Ser Leu Ser 1 5 10 Ala Ser Val Gly Glu Thr Val Thr Ile ThrCys Arg Ala Ser Gly Asn 15 20 25 Ile His Asn Tyr Leu Ala Trp Tyr Gln GlnLys Gln Gly Lys Ser Pro 30 35 40 Gln Leu Leu Val Tyr Asn Ala Lys Thr LeuAla Asp Gly Val Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly ThrGln Phe Ser Leu Lys Ile Asn 65 70 75 Ser Leu Gln Pro Glu Asp Phe Gly SerHis Tyr Cys Gln His Phe Trp 80 85 90 Thr Thr Pro Trp Thr Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 95 100 105 10 414 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 10 atg gmt tgg gtg tgg amc ttg cta ttc ctgatg gca gct gcc caa agt 48 Met Xaa Trp Val Trp Xaa Leu Leu Phe Leu MetAla Ala Ala Gln Ser -15 -10 -5 ctc caa gca cag atc cag ttg gtg cag tctgga cct gag ctg aag aag 96 Leu Gln Ala Gln Ile Gln Leu Val Gln Ser GlyPro Glu Leu Lys Lys 1 5 10 cct gga gaa aca gtc aag atc tcc tgc aag gcctct ggg tat acc ttc 144 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala SerGly Tyr Thr Phe 15 20 25 aca aac tat gga atg aac tgg gtg aag cag gct ccagga aag ggt tta 192 Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro GlyLys Gly Leu 30 35 40 45 aag tgg atg ggc tgg ata aac acc aag agt gga gagcca aca tat gct 240 Lys Trp Met Gly Trp Ile Asn Thr Lys Ser Gly Glu ProThr Tyr Ala 50 55 60 gaa gag ttc aag gga cgg ttt gtc ttc tct ttg gaa acctct gcc agc 288 Glu Glu Phe Lys Gly Arg Phe Val Phe Ser Leu Glu Thr SerAla Ser 65 70 75 act gcc cat ttg cag atc aag aat ttc aga aat gag gac acggct aca 336 Thr Ala His Leu Gln Ile Lys Asn Phe Arg Asn Glu Asp Thr AlaThr 80 85 90 tat ttc tgt gca aga tgg gta cct ggg acc tat gct atg gac tactgg 384 Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp95 100 105 ggt caa gga acc tca gtc acc gtc tcc tca 414 Gly Gln Gly ThrSer Val Thr Val Ser Ser 110 115 11 138 PRT Artificial Sequence Alpha-2Antiplasmin Antibody 11 Met Xaa Trp Val Trp Xaa Leu Leu Phe Leu Met AlaAla Ala Gln Ser -15 -10 -5 Leu Gln Ala Gln Ile Gln Leu Val Gln Ser GlyPro Glu Leu Lys Lys 1 5 10 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys AlaSer Gly Tyr Thr Phe 15 20 25 Thr Asn Tyr Gly Met Asn Trp Val Lys Gln AlaPro Gly Lys Gly Leu 30 35 40 45 Lys Trp Met Gly Trp Ile Asn Thr Lys SerGly Glu Pro Thr Tyr Ala 50 55 60 Glu Glu Phe Lys Gly Arg Phe Val Phe SerLeu Glu Thr Ser Ala Ser 65 70 75 Thr Ala His Leu Gln Ile Lys Asn Phe ArgAsn Glu Asp Thr Ala Thr 80 85 90 Tyr Phe Cys Ala Arg Trp Val Pro Gly ThrTyr Ala Met Asp Tyr Trp 95 100 105 Gly Gln Gly Thr Ser Val Thr Val SerSer 110 115 12 414 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody12 atg gmt tgg gtg tgg amc ttg cta ttc ctg atg gca gct gcc caa agt 48Met Xaa Trp Val Trp Xaa Leu Leu Phe Leu Met Ala Ala Ala Gln Ser -15 -10-5 atc caa gca cag atc cag ttg gtg cag tct gga cct gag ctg aag aag 96Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 1 5 10cct gga gag aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc 144 ProGly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 acaaag tat gga atg aac tgg gtg aag cag gct cca gga aag ggt tta 192 Thr LysTyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 30 35 40 45 aagtgg atg ggc tgg ata aac acc aac agt gga gag cca aca tat gct 240 Lys TrpMet Gly Trp Ile Asn Thr Asn Ser Gly Glu Pro Thr Tyr Ala 50 55 60 gaa gagttc aag gga cgg ttt gcc ttc tct ttg gaa acc tct gcc agc 288 Glu Glu PheLys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 65 70 75 act gcc tatttg cag atc aac aac ctc aaa aat gag gac tcg gct aca 336 Thr Ala Tyr LeuGln Ile Asn Asn Leu Lys Asn Glu Asp Ser Ala Thr 80 85 90 tat ttc tgt gcaaga tgg gta cct ggg acc tat gct atg gac tac tgg 384 Tyr Phe Cys Ala ArgTrp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp 95 100 105 ggt caa gga acctca gtc acc gtc tcc tca 414 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 110115 13 138 PRT Artificial Sequence Alpha-2 Antiplasmin Antibody 13 MetXaa Trp Val Trp Xaa Leu Leu Phe Leu Met Ala Ala Ala Gln Ser -15 -10 -5Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 1 5 10Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25Thr Lys Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 30 35 4045 Lys Trp Met Gly Trp Ile Asn Thr Asn Ser Gly Glu Pro Thr Tyr Ala 50 5560 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 65 7075 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Ser Ala Thr 80 8590 Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp 95100 105 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 110 115 14 414 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 14 atg gmt tgg gtg tggamc ttg cta ttc ctg atg gca gct gcc caa agt 48 Met Xaa Trp Val Trp XaaLeu Leu Phe Leu Met Ala Ala Ala Gln Ser -15 -10 -5 atc caa gca cag atccag ttg gtg cag tct gga cct gag ctg aag aag 96 Ile Gln Ala Gln Ile GlnLeu Val Gln Ser Gly Pro Glu Leu Lys Lys 1 5 10 cct gga gaa aca gtc aagatc tcc tgc aag gct tct ggg tat acc ttc 144 Pro Gly Glu Thr Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 aca aac tat gga atg aac tgggtg aag cag gct cca gga aag ggt tta 192 Thr Asn Tyr Gly Met Asn Trp ValLys Gln Ala Pro Gly Lys Gly Leu 30 35 40 45 aag tgg atg ggc tgg ata aacacc aag agt gga gag cca aca tat gct 240 Lys Trp Met Gly Trp Ile Asn ThrLys Ser Gly Glu Pro Thr Tyr Ala 50 55 60 gaa gag ttc aag gga cgg ttt gccttc tct ttg gaa acc tct gcc agc 288 Glu Glu Phe Lys Gly Arg Phe Ala PheSer Leu Glu Thr Ser Ala Ser 65 70 75 act gcc aat ttg cag atc aag aac ctcaaa aat gag gac acg gct aca 336 Thr Ala Asn Leu Gln Ile Lys Asn Leu LysAsn Glu Asp Thr Ala Thr 80 85 90 tat ttc tgt gca aga tgg gta cct ggg acctat gcc atg gac tac tgg 384 Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr TyrAla Met Asp Tyr Trp 95 100 105 ggt caa gga acc tca gtc acc gtc tcc tca414 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 110 115 15 138 PRTArtificial Sequence Alpha-2 Antiplasmin Antibody 15 Met Xaa Trp Val TrpXaa Leu Leu Phe Leu Met Ala Ala Ala Gln Ser -15 -10 -5 Ile Gln Ala GlnIle Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 1 5 10 Pro Gly Glu ThrVal Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Asn Tyr GlyMet Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 30 35 40 45 Lys Trp MetGly Trp Ile Asn Thr Lys Ser Gly Glu Pro Thr Tyr Ala 50 55 60 Glu Glu PheLys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 65 70 75 Thr Ala AsnLeu Gln Ile Lys Asn Leu Lys Asn Glu Asp Thr Ala Thr 80 85 90 Tyr Phe CysAla Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp 95 100 105 Gly GlnGly Thr Ser Val Thr Val Ser Ser 110 115 16 411 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 16 atttaaattg atatctcctt aggtctcgag atg agtgtg ctc act cag gtc ctg 54 Met Ser Val Leu Thr Gln Val Leu -20 -15 gcgttg ctg ctg ctg tgg ctt aca ggt gcc aga tgt gac atc cag atg 102 Ala LeuLeu Leu Leu Trp Leu Thr Gly Ala Arg Cys Asp Ile Gln Met -10 -5 1 act cagtct cca tcc tcc cta tct gca tct gtg gga gac aga gtc acc 150 Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 5 10 15 20 atc acatgt cga gca agt ggg aat att cac aat tat tta gca tgg tat 198 Ile Thr CysArg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala Trp Tyr 25 30 35 cag cag aaacag gga aaa tct cct caa ctc ctg gtc tat aat gca aaa 246 Gln Gln Lys GlnGly Lys Ser Pro Gln Leu Leu Val Tyr Asn Ala Lys 40 45 50 acc tta gca agtggt gtg cca tca agg ttc agt ggc agt gga tca gga 294 Thr Leu Ala Ser GlyVal Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 55 60 65 aca gat ttt act ctcacc atc agc agc ctg cag cct gaa gat ttt ggg 342 Thr Asp Phe Thr Leu ThrIle Ser Ser Leu Gln Pro Glu Asp Phe Gly 70 75 80 agt cat tac tgt caa catttt tgg acc act ccg tgg acg ttc ggt gga 390 Ser His Tyr Cys Gln His PheTrp Thr Thr Pro Trp Thr Phe Gly Gly 85 90 95 100 ggc acc aag ctg gaa atcaaa 411 Gly Thr Lys Leu Glu Ile Lys 105 17 127 PRT Artificial SequenceAlpha-2 Antiplasmin Antibody 17 Met Ser Val Leu Thr Gln Val Leu Ala LeuLeu Leu Leu Trp Leu Thr -20 -15 -10 -5 Gly Ala Arg Cys Asp Ile Gln MetThr Gln Ser Pro Ser Ser Leu Ser 1 5 10 Ala Ser Val Gly Asp Arg Val ThrIle Thr Cys Arg Ala Ser Gly Asn 15 20 25 Ile His Asn Tyr Leu Ala Trp TyrGln Gln Lys Gln Gly Lys Ser Pro 30 35 40 Gln Leu Leu Val Tyr Asn Ala LysThr Leu Ala Ser Gly Val Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 Ser Leu Gln Pro Glu Asp PheGly Ser His Tyr Cys Gln His Phe Trp 80 85 90 Thr Thr Pro Trp Thr Phe GlyGly Gly Thr Lys Leu Glu Ile Lys 95 100 105 18 417 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 18 atg agt gtg ctc act cag gtc ctggcg ttg ctg ctg ctg tgg ctt aca 48 Met Ser Val Leu Thr Gln Val Leu AlaLeu Leu Leu Leu Trp Leu Thr -20 -15 -10 -5 ggt gcc aga tgt cag atc cagttg gtg cag tct gga tct gag ctg aag 96 Gly Ala Arg Cys Gln Ile Gln LeuVal Gln Ser Gly Ser Glu Leu Lys 1 5 10 aag cct gga gcc tca gtc aag atctcc tgc aag gct tct ggg tat acc 144 Lys Pro Gly Ala Ser Val Lys Ile SerCys Lys Ala Ser Gly Tyr Thr 15 20 25 ttc aca aac tat gga atg aac tgg gtgcga cag gct cca gga caa ggt 192 Phe Thr Asn Tyr Gly Met Asn Trp Val ArgGln Ala Pro Gly Gln Gly 30 35 40 tta gag tgg atg ggc tgg ata aac acc aagagt gga gag cca aca tat 240 Leu Glu Trp Met Gly Trp Ile Asn Thr Lys SerGly Glu Pro Thr Tyr 45 50 55 60 gct gaa gag ttc aag gga cgg ttt gtc ttctct ttg gac acc tct gtc 288 Ala Glu Glu Phe Lys Gly Arg Phe Val Phe SerLeu Asp Thr Ser Val 65 70 75 acc act gcc tat ttg cag atc agc agc ctc aaagct gag gac acg gct 336 Thr Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys AlaGlu Asp Thr Ala 80 85 90 gtg tat ttc tgt gca aga tgg gta cct ggg acc tatgcc atg gac tac 384 Val Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr AlaMet Asp Tyr 95 100 105 tgg ggt caa gga acc acg gtc acc gtc tcc tca 417Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 19 139 PRTArtificial Sequence Alpha-2 Antiplasmin Antibody 19 Met Ser Val Leu ThrGln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr -20 -15 -10 -5 Gly Ala ArgCys Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys 1 5 10 Lys Pro GlyAla Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr 15 20 25 Phe Thr AsnTyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly 30 35 40 Leu Glu TrpMet Gly Trp Ile Asn Thr Lys Ser Gly Glu Pro Thr Tyr 45 50 55 60 Ala GluGlu Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val 65 70 75 Thr ThrAla Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala 80 85 90 Val TyrPhe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr 95 100 105 TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 110 115 20 447 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 20 cgattggaat tcttgcggccgcttgctagc atg agt gtg ctc act cag gtc ctg 54 Met Ser Val Leu Thr GlnVal Leu -20 -15 gcg ttg ctg ctg ctg tgg ctt aca ggt gcc aga tgt cag atccag ttg 102 Ala Leu Leu Leu Leu Trp Leu Thr Gly Ala Arg Cys Gln Ile GlnLeu -10 -5 1 gtg cag tct gga gct gag gtg aag aag cct gga gcc tca gtc aagatc 150 Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Ile5 10 15 20 tcc tgc aag gct tct ggg tat acc ttc aca aac tat gga atg aactgg 198 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp25 30 35 gtg cga cag gct cca gga caa ggt tta gag tgg atg ggc tgg ata aac246 Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn 4045 50 acc aag agt gga gag cca aca tat gct gaa gag ttc aag gga cgg ttt294 Thr Lys Ser Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg Phe 5560 65 acc ttc acc ttg gac acc tct acg agc act gcc tat ttg gag atc agg342 Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr Ala Tyr Leu Glu Ile Arg 7075 80 agc ctc aga tct gac gac acg gct gtg tat ttc tgt gca aga tgg gta390 Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Trp Val 8590 95 100 cct ggg acc tat gcc atg gac tac tgg ggt caa gga acc acg gtcacc 438 Pro Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr105 110 115 gtc tcc tca 447 Val Ser Ser 21 139 PRT Artificial SequenceAlpha-2 Antiplasmin Antibody 21 Met Ser Val Leu Thr Gln Val Leu Ala LeuLeu Leu Leu Trp Leu Thr -20 -15 -10 -5 Gly Ala Arg Cys Gln Ile Gln LeuVal Gln Ser Gly Ala Glu Val Lys 1 5 10 Lys Pro Gly Ala Ser Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr 15 20 25 Phe Thr Asn Tyr Gly Met Asn TrpVal Arg Gln Ala Pro Gly Gln Gly 30 35 40 Leu Glu Trp Met Gly Trp Ile AsnThr Lys Ser Gly Glu Pro Thr Tyr 45 50 55 60 Ala Glu Glu Phe Lys Gly ArgPhe Thr Phe Thr Leu Asp Thr Ser Thr 65 70 75 Ser Thr Ala Tyr Leu Glu IleArg Ser Leu Arg Ser Asp Asp Thr Ala 80 85 90 Val Tyr Phe Cys Ala Arg TrpVal Pro Gly Thr Tyr Ala Met Asp Tyr 95 100 105 Trp Gly Gln Gly Thr ThrVal Thr Val Ser Ser 110 115 22 33 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 22 nnnnnngaat tcactggatg gtgggaagat gga 33 23 42DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 23 nnnnnngaattcayctccac acacaggrrc cagtggatag ac 42 24 40 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 24 actagtcgac atgagtgtgc tcactcaggtcctggsgttg 40 25 88 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody25 tagggagacc caagcttggt accaatttaa attgatatct ccttaggtct cgagtctcta 60gataaccggt caatcgattg ggattctt 88 26 88 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 26 gacactatag aatagggccc ttccgcggtt ggatccaacacgtgaagcta gcaagcggcc 60 gcaagaattc caatcgattg accggtta 88 27 41 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 27 gatctgctagcccgggtgac ctgaggcgcg cctttggcgc c 41 28 41 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 28 gatcggcgcc aaaggcgcgc cgcaggtcacccgggctagc a 41 29 32 DNA Artificial Sequence Alpha-2 AntiplasminAntibody 29 ccgggcctct caaaaaaggg aaaaaaagca tg 32 30 24 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 30 ctttttttcc cttttttgag aggc 2431 74 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 31 cgcgccggcttcgaatagcc agagtaacct ttttttttaa ttttatttta ttttattttt 60 gagatggagtttgg 74 32 72 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 32cgccaaactc catctcaaaa ataaaataaa ataaaattaa aaaaaaaggt tactctggct 60attcgaagcc gg 72 33 24 DNA Artificial Sequence Alpha-2 AntiplasminAntibody 33 atcgatgcta gcaccaaggg ccca 24 34 24 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 34 ctcgaggggt caccacgctg ctga 24 35 21 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 35 aacagctatgaccatgatta c 21 36 21 DNA Artificial Sequence Alpha-2 AntiplasminAntibody 36 cacccagcct gtgcctgcct g 21 37 30 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 37 cgattggaat tcttgcggcc gcttgctagc 30 3880 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 38 cttgcggccgcttgctagca tggattgggt gtggaacttg ctattcctga tggcagctgc 60 ccaaagtatccaagcacaga 80 39 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody39 cttgactgtt tctccaggct tcttcagctc aggtccagac tgcaccaact ggatctgtgc 60ttggatactt tgggcagctg 80 40 80 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 40 ctgaagaagc ctggagaaac agtcaagatc tcctgcaaggcttctgggta taccttcaca 60 aactatggaa tgaactgggt 80 41 80 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 41 tcttggtgtt tatccagcccatccacttta aaccctttcc tggagcctgc ttcacccagt 60 tcattccata gtttgtgaag 8042 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 42 agtggatgggctggataaac accaagagtg gagagccaac atatgctgaa gagttcaagg 60 gacggtttgccttctctttg 80 43 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody43 tcctcatttt tgaggttctt gatctgcaaa ttggcagtgc tggcagaggt ttccaaagag 60aaggcaaacc gtcccttgaa 80 44 80 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 44 gcagatcaag aacctcaaaa atgaggacac ggctacatatttctgtgcaa gatgggtacc 60 tgggacctat gccatggact 80 45 80 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 45 tgggcccttg gtgctagctgaggagacggt gactgaggtt ccttgacccc agtagtccat 60 ggcataggtc ccaggtaccc 8046 29 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 46 gggaagacggatgggccctt ggtgctagc 29 47 30 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 47 atttaaattg atatctcctt aggtctcgag 30 48 79 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 48 atttaaattgatatctcctt aggtctcgag atgagtgtgc tcactcaggt cctggcgttg 60 ctgctgctgtggcttacag 79 49 78 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody49 agatgcagat agggaggctg gagactgagt catctggatg tcacatctgg cacctgtaag 60ccacagcagc agcaacgc 78 50 78 DNA Artificial Sequence Alpha-2 AntiplasminAntibody 50 gtctccagcc tccctatctg catctgtggg agaaactgtc accatcacatgtcgagcaag 60 tgggaatatt cacaatta 78 51 78 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 51 tatagaccag gagctgagga gattttccctgtttctgctg ataccatgct aaataattgt 60 gaatattccc acttgctc 78 52 78 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 52 aaatctcctcagctcctggt ctataatgca aaaaccttag cagatggtgt gccatcaagg 60 ttcagtggcagtggatca 78 53 78 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody53 ctcccaaaat cttcaggctg caggctgttg atcctgagag aaaattgtgt tcctgatcca 60ctgccactga accttgat 78 54 78 DNA Artificial Sequence Alpha-2 AntiplasminAntibody 54 gcctgcagcc tgaagatttt gggagtcatt actgtcaaca tttttggaccactccgtgga 60 cgttcggtgg aggcacca 78 55 81 DNA Artificial SequenceAlpha-2 Antiplasmin Antibody 55 ttccaatcga ttgaccggtt atctagagactcgagactta cgtttgattt ccagcttggt 60 gcctccaccg aacgtccacg g 81 56 30 DNAArtificial Sequence Alpha-2 Antiplasmin Antibody 56 tcgattgaccggttatctag agactcgaga 30 57 80 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 57 cttgcggccg cttgctagca tgagtgtgct cactcaggtcctggcgttgc tgctgctgtg 60 gcttacaggt gccagatgtc 80 58 80 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 58 gactgaggct ccaggcttcttcagctcaga tccagactgc accaactgga tctgacatct 60 ggcacctgta agccacagca 8059 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 59 gagctgaagaagcctggagc ctcagtcaag atctcctgca aggcttctgg gtataccttc 60 acaaactatggaatgaactg 80 60 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody60 tggtgtttat ccagcccatc cactctaaac cttgtcctgg agcctgtcgc acccagttca 60ttccatagtt tgtgaaggta 80 61 80 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 61 tagagtggat gggctggata aacaccaaga gtggagagccaacatatgct gaagagttca 60 agggacggtt tgtcttctct 80 62 80 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 62 tcagctttga ggctgctgatctgcaaatag gcagtgctga cagaggtgtc caaagagaag 60 acaaaccgtc ccttgaactc 8063 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 63 tttgcagatcagcagcctca aagctgagga cacggctgtg tatttctgtg caagatgggt 60 acctgggacctatgccatgg 80 64 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody64 gcccttggtg ctagctgagg agacggtgac cgtggttcct tgaccccagt agtccatggc 60ataggtccca ggtacccatc 80 65 80 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 65 tgctgtggct tacaggtgcc agatgtcaga tccagttggtgcagtctgga gctgaggtga 60 agaagcctgg agcctcagtc 80 66 80 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 66 tagagtggat gggctggataaacaccaaga gtggagagcc aacatatgct gaagagttca 60 agggacggtt taccttcacc 8067 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 67 tcagatctgaggctcctgat ctccaaatag gcagtgctcg tagaggtgtc caaggtgaag 60 gtaaaccgtcccttgaactc 80 68 80 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody68 tttggagatc aggagcctca gatctgacga cacggctgtg tatttctgtg caagatgggt 60acctgggacc tatgccatgg 80 69 78 DNA Artificial Sequence Alpha-2Antiplasmin Antibody 69 agatgcagat agggaggatg gagactgagt catctggatgtcacatctgg cacctgtaag 60 ccacagcagc agcaacgc 78 70 78 DNA ArtificialSequence Alpha-2 Antiplasmin Antibody 70 gtctccatcc tccctatctgcatctgtggg agacagagtc accatcacat gtcgagcaag 60 tgggaatatt cacaatta 78 7178 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody 71 aaatctcctcaactcctggt ctataatgca aaaaccttag caagtggtgt gccatcaagg 60 ttcagtggcagtggatca 78 72 78 DNA Artificial Sequence Alpha-2 Antiplasmin Antibody72 ctcccaaaat cttcaggctg caggctgctg atggtgagag taaaatctgt tcctgatcca 60ctgccactga accttgat 78 73 18 PRT Artificial Sequence Alpha-2 AntiplasminAntibody 73 Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser Pro SerLeu 1 5 10 15 Thr Thr 74 15 PRT Artificial Sequence Alpha-2 AntiplasminAntibody 74 Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser1 5 10 15 75 107 PRT Artificial Sequence Alpha-2 Antiplasmin Antibody 75Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 1015 Glu Thr Val Thr Xaa Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 2530 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 4045 Tyr Asn Ala Xaa Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 5560 Ser Gly Ser Gly Thr Gln Xaa Ser Leu Xaa Ile Asn Ser Leu Gln Pro 65 7075 80 Glu Asp Phe Gly Ser Xaa Tyr Cys Gln His Phe Trp Xaa Xaa Pro Trp 8590 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 76 107 PRTArtificial Sequence Alpha-2 Antiplasmin Antibody 76 Asp Ile Gln Met ThrGln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val ThrIle Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala Trp TyrGln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala LysThr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser GlyThr Gln Phe Ser Leu Xaa Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp PheGly Ser His Tyr Cys Gln His Phe Trp Thr Thr Pro Trp 85 90 95 Thr Phe GlyGly Gly Thr Lys Leu Glu Ile Lys 100 105 77 107 PRT Artificial SequenceAlpha-2 Antiplasmin Antibody 77 Asp Ile Gln Met Thr Gln Ser Pro Xaa SerLeu Ser Ala Ser Val Gly 1 5 10 15 Xaa Xaa Val Thr Xaa Thr Cys Arg AlaSer Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln GlyLys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Xaa Thr Leu Ala Xaa GlyVal Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Xaa Xaa Xaa LeuXaa Ile Xaa Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Xaa Tyr CysGln His Phe Trp Xaa Xaa Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys LeuGlu Ile Lys 100 105 78 119 PRT Artificial Sequence Alpha-2 AntiplasminAntibody 78 Gln Ile Gln Leu Val Gln Ser Gly Xaa Glu Xaa Lys Lys Pro GlyAla 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe ThrAsn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met 35 40 45 Gly Trp Ile Asn Thr Lys Ser Gly Glu Pro Thr Tyr Ala GluGlu Phe 50 55 60 Lys Gly Arg Phe Xaa Phe Xaa Leu Asp Thr Ser Xaa Ser ThrAla Tyr 65 70 75 80 Leu Xaa Ile Xaa Ser Leu Xaa Xaa Xaa Asp Thr Ala ValTyr Phe Cys 85 90 95 Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr TrpGly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 79 119 PRTArtificial Sequence Alpha-2 Antiplasmin Antibody 79 Gln Ile Gln Leu ValGln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys IleSer Cys Xaa Ala Ser Gly Tyr Thr Phe Thr Xaa Tyr 20 25 30 Gly Met Asn TrpVal Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile AsnThr Xaa Ser Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg PheXaa Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Xaa 65 70 75 80 Leu Gln IleXaa Asn Xaa Xaa Asn Glu Asp Xaa Ala Thr Tyr Phe Cys 85 90 95 Ala Arg TrpVal Pro Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr SerVal Thr Val Ser Ser 115 80 119 PRT Artificial Sequence Alpha-2Antiplasmin Antibody 80 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu LysLys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Xaa Ala Ser Gly TyrThr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly LysGly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Lys Ser Gly Glu Pro ThrTyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Xaa Phe Ser Leu Glu Thr SerAla Ser Thr Ala Xaa 65 70 75 80 Leu Gln Ile Lys Asn Xaa Xaa Asn Glu AspThr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Trp Val Pro Gly Thr Tyr Ala MetAsp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 81119 PRT Artificial Sequence Alpha-2 Antiplasmin Antibody 81 Gln Ile GlnLeu Val Gln Ser Gly Xaa Glu Xaa Lys Lys Pro Gly Xaa 1 5 10 15 Xaa ValLys Ile Ser Cys Xaa Ala Ser Gly Tyr Thr Phe Thr Xaa Tyr 20 25 30 Gly MetAsn Trp Val Xaa Gln Ala Pro Gly Xaa Gly Leu Xaa Trp Met 35 40 45 Gly TrpIle Asn Thr Xaa Ser Gly Glu Pro Thr Tyr Ala Glu Glu Phe 50 55 60 Lys GlyArg Phe Xaa Phe Xaa Leu Xaa Thr Ser Xaa Ser Thr Ala Xaa 65 70 75 80 LeuXaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Ala Xaa Tyr Phe Cys 85 90 95 AlaArg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Xaa Val Thr Val Ser Ser 115

What is claimed is:
 1. An immunologic molecule wherein said immunologicmolecule is capable of binding to both (1) human and nonhumancirculating α2-antiplasmins and (2) human and nonhuman fibrincrosslinked α2-antiplasmins.
 2. The immunologic molecule of claim 1,wherein said immunologic molecule is a chimeric antibody.
 3. Theimmunologic molecule of claim 1, wherein said immunologic molecule is ahumanized antibody.
 4. The immunologic molecule of claim 1, wherein saidimmunologic molecule is an antibody fragment.
 5. The immunologicmolecule of claim 1, wherein said immunologic molecule is a monoclonalantibody.
 6. The immunologic molecule of claim 1, wherein saidimmunologic molecule comprises amino acids 1 to 107 of SEQ ID NO: 9 andamino acids 1 to 119 of SEQ ID NO:
 15. 7. The immunologic molecule ofclaim 1, wherein said immunologic molecule comprises amino acids 1 to107 of SEQ ID NO: 5 and amino acids 1 to 119 of SEQ ID NO:
 11. 8. Theimmunologic molecule of claim 1, wherein said immunologic moleculecomprises amino acids 1 to 107 of SEQ ID NO: 7 and amino acids 1 to119of SEQ ID NO:
 13. 9. The immunologic molecule of claim 1, selectedfrom the group consisting of: (a) an immunologic molecule, wherein theCDR1 region of the light chain of said immunologic molecule comprisesamino acids 26 to 32 of SEQ ID NO: 75; (b) an immunologic molecule,wherein the CDR2 region of the light chain of said immunologic moleculecomprises amino acids 50 to 52 of SEQ ID NO: 75; (c) an immunologicmolecule, wherein the CDR3 region of the light chain of said immunologicmolecule comprises amino acids 91 to 96 of SEQ ID NO: 75; (d) animmunologic molecule, wherein the CDR1 region of the heavy chain of saidimmunologic molecule comprises amino acids 26 to 32 of SEQ ID NO: 79;(e) an immunologic molecule, wherein the CDR2 region of the heavy chainof said immunologic molecule comprises amino acids 53 to 56 of SEQ IDNO: 79; and (f) an immunologic molecule, wherein the CDR3 region of theheavy chain of said immunologic molecule comprises amino acids 100 to107 of SEQ ID NO:
 79. 10. The immunologic molecule of claim 1, selectedfrom the group consisting of: (a) an immunologic molecule, wherein theCDR1 region of the light chain of said immunologic molecule comprisesamino acids 26 to 32 of SEQ ID NO: 76; (b) an immunologic molecule,wherein the CDR2 region of the light chain of said immunologic moleculecomprises amino acids 50 to 52 of SEQ ID NO: 76; (c) an immunologicmolecule, wherein the CDR3 region of the light chain of said immunologicmolecule comprises amino acids 91 to 96 of SEQ ID NO: 76; (d) animmunologic molecule, wherein the CDR1 region of the heavy chain of saidimmunologic molecule comprises amino acids 26 to 32 of SEQ ID NO: 80;(e) an immunologic molecule, wherein the CDR2 region of the heavy chainof said immunologic molecule comprises amino acids 53 to 56 of SEQ IDNO: 80; and (f) an immunologic molecule, wherein the CDR3 region of theheavy chain of said immunologic molecule comprises amino acids 100 to107 of SEQ ID NO:
 80. 11. The monoclonal antibody of claim 5, whereinsaid monoclonal antibody is 77A3.
 12. The monoclonal antibody of claim5, wherein said monoclonal antibody is 49C9.
 13. The monoclonal antibodyof claim 5, wherein said monoclonal antibody is 70B11.
 14. A method ofmaking the monoclonal antibody of claim 5 comprising: (a) immunizing ananimal with α2-antiplasmin or fragment thereof; (b) fusing cells fromthe animal with tumor cells to make a hybridoma cell line; (c) cloningthe hybridoma cell line; (d) selecting for the monoclonal antibodycapable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins; and (e) obtaining the monoclonal antibody.
 15. Ahybridoma cell line which produces the monoclonal antibody of claim 5.16. The hybridoma cell line of claim 15, wherein said hybridoma cellline is ATCC Accession No. HB-12192.
 17. A method of making thehybridoma cell line of claim 15 comprising: (a) immunizing an animalwith α2-antiplasmin or fragment thereof; (b) fusing the cells from theanimal with tumor cells to make the hybridoma cell line; and (c)obtaining the hybridoma cell line which produces the monoclonal antibodycapable of binding to both (1) human and nonhuman circulatingα2-antiplasmins and (2) human and nonhuman fibrin crosslinkedα2-antiplasmins.
 18. A nucleic acid molecule, selected from the groupconsisting of: (a) a nucleic acid molecule comprising a nucleotidesequence encoding for amino acids 1 to 107 of SEQ ID NO: 5; (b) anucleic acid molecule comprising a nucleotide sequence encoding foramino acids 1 to 107 of SEQ ID NO: 7; (c) a nucleic acid moleculecomprising a nucleotide sequence encoding for amino acids 1 to 107 ofSEQ ID NO: 9; (d) a nucleic acid molecule comprising a nucleotidesequence encoding for amino acids 1 to 107 of SEQ ID NO: 75; (e) anucleic acid molecule comprising a nucleotide sequence encoding foramino acids 1 to 119 of SEQ ID NO: 11; (f) a nucleic acid moleculecomprising a nucleotide sequence encoding for amino acids 1 to 119 ofSEQ ID NO: 13; (g) a nucleic acid molecule comprising a nucleotidesequence encoding for amino acids 1 to 119 of SEQ ID NO: 15; and (h) anucleic acid molecule comprising a nucleotide sequence encoding foramino acids 1 to 119 of SEQ ID NO:
 79. 19. A method for treatingpulmonary embolism, myocardial infarction, or thrombosis in a patientcomprising administering a therapeutically effective amount of animmunologic molecule of claim 1 to said patient.
 20. The method of claim19, wherein said immunologic molecule is a monoclonal antibody.
 21. Themethod of claim 20, wherein said monoclonal antibody is 77A3.
 22. Themethod of claim 19, wherein said immunologic molecule is administered bycontinuous intravenous infusion or by bolus.
 23. A method of treatmentfor pulmonary embolism, myocardial infarction, or thrombosis in apatient which comprises co-administering to a patient in need of suchtreatment: (a) a therapeutically effective amount of an immunologicmolecule of claim 1; and (b) a therapeutically effective amount of athrombolytic agent, wherein said immunologic molecule (a) is differentfrom said thrombolytic agent (b), thereby treating said patient.
 24. Themethod of claim 23, wherein said immunologic molecule is a monoclonalantibody.
 25. The method of claim 24, wherein said monoclonal antibodyis 77A3.
 26. The method of claim 23, wherein said thrombolytic agent isplasmin.
 27. The method of claim 23, wherein said thrombolytic agent isan anti-coagulant which inhibits fibrin.
 28. The method of claim 27,wherein said anti-coagulant is selected from the group consisting ofheparin, hirudin and activated protein C.
 29. The method of claim 23,wherein said thrombolytic agent is an anti-coagulant which inhibitsplatelets.
 30. The method of claim 23, wherein said thrombolytic agentis a plasminogen activator.
 31. The method of claim 30, wherein saidplasminogen activator is selected from the group consisting ofstreptokinase, prourokinase, urokinase, tissue-type plasminogenactivator, staphylokinase, and vampire bat plasminogen activator. 32.The method of claim 23, wherein both said immunologic molecule (a) andsaid thrombolytic agent (b) are provided to said patient by anintravenous infusion or by an intravenously injected bolus.
 33. Themethod of claim 23, wherein said patient is provided with a first boluscontaining said immunologic molecule (a) and a subsequently administeredsecond bolus containing said thrombolytic agent (b).
 34. The method ofclaim 23, wherein: (1) said immunologic molecule (a) is provided to saidpatient at a dose of between 3 to 600 nmole per kg of patient weight;and (2) said thrombolytic agent (b) is provided to said patient at adose of between 0.01 to 3.0 mg per kg of patient weight.
 35. A kituseful for carrying out the method of claim 23, being compartmentalizedin close confinement to receive two or more container means therein,which comprises: (1) a first container containing a therapeuticallyeffective amount of said immunologic molecule (a); and (2) a secondcontainer containing a therapeutically effective amount of saidthrombolytic agent (b), wherein said immunologic molecule (a) isdifferent from said thrombolytic agent (b).